Featured DDDDK tag antibodies

DDDDK tag is a polypeptide protein tag that can be added to a protein using recombinant DNA technology, which allows elution under non-denaturing conditions. The DDDDK tag is likely to be located on the surface of a fusion protein because of its hydrophilic nature, and therefore is more likely to be accessible to antibodies. It not only has been used for studying proteins in living cells and for protein purifcation by affinity chromatography, but also has been used in the isolation of protein complexes with multiple subunits.

Abbkine offers a full range of anti-DDDDK tag antibodies for your choice. The antibodies not only include highly specific monoclonal and polyclonal DDDDK tag antibodies, but also contain AbFluor™ (350, 405, 488, 555, 594, 647, 680) dyes, Cy3, Cy5, FITC, agarose, magnetic beads, HRP conjugated anti-DDDDK tag antibodies.

[table id=16 /]

About Abbkine Scientific Co., Ltd.

Abbkine Scientific Co., Ltd. is a leading biotechnology company that focuses on developing and providing innovative, high quality assay kits, recombinant proteins, antibodies and other research tools to accelerate life science fundamental research, drug discovery, etc.  Find more details, please visit the website at Abbkine.

More epitope tag antibodies to be continued...

The Nobel Prize in Physiology or Medicine 2011

The Nobel Prize in Physiology or Medicine 2011 was divided, one half jointly to Bruce A. Beutler and Jules A. Hoffmann "for their discoveries concerning the activation of innate immunity" and the other half to Ralph M. Steinman "for his discovery of the dendritic cell and its role in adaptive immunity".

Nobelist	Born	Affiliation at the time of the award
Bruce A. Beutler	29 December 1957, Chicago, IL, USA	University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA, The Scripps Research Institute, La Jolla, CA, USA
Jules A. Hoffmann	2 August 1941, Echternach, Luxembourg	University of Strasbourg, Strasbourg, France
Ralph M. Steinman	Born: 14 January 1943, Montreal, Canada Died: 30 September 2011	Rockefeller University, New York, NY, USA

Summary

This year's Nobel Laureates have revolutionized our understanding of the immune system by discovering key principles for its activation.

Scientists have long been searching for the gatekeepers of the immune response by which man and other animals defend themselves against attack by bacteria and other microorganisms. Bruce Beutler and Jules Hoffmann discovered receptor proteins that can recognize such microorganisms and activate innate immunity, the first step in the body's immune response. Ralph Steinman discovered the dendritic cells of the immune system and their unique capacity to activate and regulate adaptive immunity, the later stage of the immune response during which microorganisms are cleared from the body.

The discoveries of the three Nobel Laureates have revealed how the innate and adaptive phases of the immune response are activated and thereby provided novel insights into disease mechanisms. Their work has opened up new avenues for the development of prevention and therapy against infections, cancer, and inflammatory diseases.

More details, please click The 2011 Nobel Prize in Physiology or Medicine.

The Nobel Prize in Physiology or Medicine 2011

The Nobel Prize in Physiology or Medicine 2011 was divided, one half jointly to Bruce A. Beutler and Jules A. Hoffmann "for their discoveries concerning the activation of innate immunity" and the other half to Ralph M. Steinman "for his discovery of the dendritic cell and its role in adaptive immunity".

Nobelist	Born	Affiliation at the time of the award
Bruce A. Beutler	29 December 1957, Chicago, IL, USA	University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA, The Scripps Research Institute, La Jolla, CA, USA
Jules A. Hoffmann	2 August 1941, Echternach, Luxembourg	University of Strasbourg, Strasbourg, France
Ralph M. Steinman	Born: 14 January 1943, Montreal, Canada Died: 30 September 2011	Rockefeller University, New York, NY, USA

Summary

This year's Nobel Laureates have revolutionized our understanding of the immune system by discovering key principles for its activation.

Scientists have long been searching for the gatekeepers of the immune response by which man and other animals defend themselves against attack by bacteria and other microorganisms. Bruce Beutler and Jules Hoffmann discovered receptor proteins that can recognize such microorganisms and activate innate immunity, the first step in the body's immune response. Ralph Steinman discovered the dendritic cells of the immune system and their unique capacity to activate and regulate adaptive immunity, the later stage of the immune response during which microorganisms are cleared from the body.

The discoveries of the three Nobel Laureates have revealed how the innate and adaptive phases of the immune response are activated and thereby provided novel insights into disease mechanisms. Their work has opened up new avenues for the development of prevention and therapy against infections, cancer, and inflammatory diseases.

More details, please click The 2011 Nobel Prize in Physiology or Medicine.

Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass

Content introduction:

• Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass


• De novo DNA synthesis using polymerase-nucleotide conjugates


• Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos


• Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER


• Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions

1. Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass
Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia–associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, Ruwan Gunaratne at Duke University in Durham, North Carolina, USA and his colleagues solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. They demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer–drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Their results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.



Read more, please click https://www.nature.com/articles/nbt.4153

2. De novo DNA synthesis using polymerase-nucleotide conjugates
Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, Sebastian Palluk at Joint BioEnergy Institute in California, USA and his colleagues describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3′ end of the primer remains covalently bound to TdT and is inaccessible to other TdT–dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. They demonstrate that TdT–dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10–20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

Read more, please click https://www.nature.com/articles/nbt.4173

3. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos
Rapid, efficient generation of knock-in mice with targeted large insertions remains a major hurdle in mouse genetics. Here, Bin Gu at Hospital for Sick Children in Toronto, Ontario, Canada and his colleagues describe two-cell homologous recombination (2C-HR)-CRISPR, a highly efficient gene-editing method based on introducing CRISPR reagents into embryos at the two-cell stage, which takes advantage of the open chromatin structure and the likely increase in homologous-recombination efficiency during the long G2 phase. Combining 2C-HR-CRISPR with a modified biotin–streptavidin approach to localize repair templates to target sites, they achieved a more-than-tenfold increase (up to 95%) in knock-in efficiency over standard methods. They targeted 20 endogenous genes expressed in blastocysts with fluorescent reporters and generated reporter mouse lines. They also generated triple-color blastocysts with all three lineages differentially labeled, as well as embryos carrying the two-component auxin-inducible degradation system for probing protein function. They suggest that 2C-HR-CRISPR is superior to random transgenesis or standard genome-editing protocols, because it ensures highly efficient insertions at endogenous loci and defined 'safe harbor' sites.

Read more, please click https://www.nature.com/articles/nbt.4166

4. Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER
Precise control over microbial cell growth conditions could enable detection of minute phenotypic changes, which would improve our understanding of how genotypes are shaped by adaptive selection. Although automated cell-culture systems such as bioreactors offer strict control over liquid culture conditions, they often do not scale to high-throughput or require cumbersome redesign to alter growth conditions. Brandon G Wong at Boston University in Boston, Massachusetts, USA and his colleagues report the design and validation of eVOLVER, a scalable do-it-yourself (DIY) framework, which can be configured to carry out high-throughput growth experiments in molecular evolution, systems biology, and microbiology. High-throughput evolution of yeast populations grown at different densities reveals that eVOLVER can be applied to characterize adaptive niches. Growth selection on a genome-wide yeast knockout library, using temperatures varied over different timescales, finds strains sensitive to temperature changes or frequency of temperature change. Inspired by large-scale integration of electronics and microfluidics, they also demonstrate millifluidic multiplexing modules that enable multiplexed media routing, cleaning, vial-to-vial transfers and automated yeast mating.

Read more, please click https://www.nature.com/articles/nbt.4151

5. Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions
Post-translational phosphorylation is essential to human cellular processes, but the transient, heterogeneous nature of this modification complicates its study in native systems. Karl W Barber at Yale University in New Haven, Connecticut, USA and his colleagues developed an approach to interrogate phosphorylation and its role in protein-protein interactions on a proteome-wide scale. They genetically encoded phosphoserine in recoded E. coli and generated a peptide-based heterologous representation of the human serine phosphoproteome. They designed a single-plasmid library encoding >100,000 human phosphopeptides and confirmed the site-specific incorporation of phosphoserine in >36,000 of these peptides. They then integrated their phosphopeptide library into an approach known as Hi-P to enable proteome-level screens for serine-phosphorylation-dependent human protein interactions. Using Hi-P, they found hundreds of known and potentially new phosphoserine-dependent interactors with 14-3-3 proteins and WW domains. These phosphosites retained important binding characteristics of the native human phosphoproteome, as determined by motif analysis and pull-downs using full-length phosphoproteins. This technology can be used to interrogate user-defined phosphoproteomes in any organism, tissue, or disease of interest.

Read more, please click https://www.nature.com/articles/nbt.4150



 

Immunoprecipitation

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or extrinsically labeled cells, subcellular fractions from either unlabeled or labeled cells, or in vitro–translated proteins. Immunoprecipitation is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or sedimentation on density gradients. Either polyclonal or monoclonal antibodies from various animal species can be used in immunoprecipitation protocols. Antibodies can be bound noncovalently to immunoadsorbents such as protein A– or protein G–agarose, or can be coupled covalently to a solid-phase matrix.

Figure 1 Schematic representation of the stages of a typical immunoprecipitation protocol

Immunoprecipitation protocols consist of several stages (Fig 1).
In stage 1, the antigen is solubilized by one of several techniques for lysing cells. Soluble and membrane-associated antigens can be released from cells grown either in suspension culture or as a monolayer on tissue culture dishes with nondenaturing detergents. Cells can also be lysed under denaturing conditions. Soluble antigens can also be extracted by mechanical disruption of cells in the absence of detergents. All of these lysis procedures are suitable for extracting antigens from animal cells. Two commonly used buffers for cell lysis: RIPA buffer gives lower background in IP. However, RIPA can denature some proteins. If you are conducting IP experiments to study protein-protein interactions, RIPA should not be used as it can disrupt the interactions; NP-40 buffer denatures proteins to a lesser extent, and is thus used for phosphorylation experiments when studying kinase activity. NP-40 is typically used for the study of protein-protein interactions. NP-40 is a nonionic detergent and the most commonly used detergent in cell lysis buffers for IP and westerns. Yeast cells require disruption of their cell wall in order to allow extraction of the antigens.

In stage 2, a specific antibody is attached, either noncovalently or covalently, to a sedimentable, solid-phase matrix to allow separation by low-speed centrifugation. For example, the noncovalent attachment of antibody to protein A or protein G agarose beads. Incubation will depend upon the concentration of target protein and the specificity of the Ab toward this target (Table 1).

Table 1 Binding characteristics of different immunoglobulins (Igs).

++, moderate to strong binding; +, weak binding; −, no binding

Stage 3 consists of incubating the solubilized antigen from stage 1 with the immobilized antibody from stage 2, followed by extensive washing to remove unbound proteins. Immunoprecipitated antigens can be dissociated from antibodies and reprecipitated by a protocol referred to as “immunoprecipitation-recapture”. This protocol can be used with the same antibody for further purification of the antigen, or with a second antibody to identify components of multisubunit complexes or to study protein-protein interactions. Immunoprecipitated antigens can be analyzed by one-dimensional electrophoresis, two-dimensional electrophoresis, or immunoblotting. In some cases, immunoprecipitates can be used for structural or functional analyses of the isolated antigens. Immunoprecipitates can also be used as sources of immunogens for production of monoclonal or polyclonal antibodies.

Overview of Cell Fractionation

Cell fractionation has enjoyed widespread use among cell biologists for half a century. It continues to be a fruitful, if not essential, approach in the reductionistic efforts to define the composition and functions of the multiple compartments in eukaryotic cells. It also provides the essential ingredients for the increasing number of cell-free assays now being used in test-tube reconstructions of complex cellular events involving intercompartmental interactions. Much of the knowledge regarding the composition and function of cell organelles has resulted from fractionation of mammalian tissues, where cells are both abundant and highly differentiated (and thus organelle-rich). However, with new techniques in molecular biology and widened interest in combining studies of intact cells and functional reconstitution, interest in fractionation has spread to cultured cell lines and genetically tractable lower eukaryotic cells.

The goals of cell fractionation often differ depending on the nature of the experiments being conducted. In preparative procedures, in which the intent is to isolate quantities of a particular cell organelle for further study or for subfractionation, the emphasis is on purity and (secondarily) on yield. In analytical experiments, in which the intent is not isolation of organelles but evaluation of associations of selected macromolecules with particular organelles, the emphasis is on using one-step procedures that result in different distributions of various organelles (as defined by marker activities) rather than on separating organelles outright. Finally, in preparing organelles for cell-free reconstitution, the goal is to maintain them in a functional state. The investigator generally has developed a specific assay for intercompartmental interaction that does not rely on organelle purity, so the extent of contamination by irrelevant organelles is less important.

The separation of distinct organelles during cell fractionation results from their differing physical properties-size (and shape), buoyant density, and surface charge density-which reflect their differing compositions. Particular fractionation techniques capitalize on one or more of these properties. For example, gel filtration separates on the basis of size, centrifugation separates on the basis of size and density, and electrophoresis separates on the basis of surface charge density. As knowledge of the specific composition of particular organelles has developed, it has become possible to apply newer techniques such as affinity chromatography and selective density-shift perturbation. Whichever fractionation method is used, the procedures may have to be modified to adapt them to individual needs. Even isolation of a particular kind of organelle from different tissue or cell sources may necessitate adjustment of fractionation conditions. This also means that in addition to isolating the organelle of interest, one should also plan on confirming the identity of what has been isolated.

Centrifugation is the most widely used procedure in cell fractionation. It is the only approach commonly used to separate crude tissue homogenates (often having quite large volumes) into subfractions as starting material for more refined purification procedures. Further, the technology available, using rotors with a variety of geometries and diverse media that enable separation according to size, density, or both, now routinely permits refined separations on volumes ranging from submilliliter to several liters. No other technology is this versatile. Gel filtration is limited by the pore sizes of available resins, such that only regularly shaped vesicles with diameters <100 to 200 nm can be purified away from larger or irregularly shaped organelles. Use of electrophoresis for organelle purification, especially at the preparative level, is relatively recent, and, as with gel filtration, its success relies on generating starting material by centrifugation. Although it shows promise for purifying selected organelles (e.g., endosomes) that have been difficult to obtain otherwise, surface charge densities on most organelles may not be different enough (or able to be manipulated sufficiently) to make this a versatile procedure.

Figure 1 The separation of distinct organelles during cell fractionation by centrifugation

Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass

Content introduction:

• Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass


• De novo DNA synthesis using polymerase-nucleotide conjugates


• Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos


• Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER


• Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions

1. Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass
Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia–associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, Ruwan Gunaratne at Duke University in Durham, North Carolina, USA and his colleagues solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. They demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer–drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Their results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.



Read more, please click https://www.nature.com/articles/nbt.4153

2. De novo DNA synthesis using polymerase-nucleotide conjugates
Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, Sebastian Palluk at Joint BioEnergy Institute in California, USA and his colleagues describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3′ end of the primer remains covalently bound to TdT and is inaccessible to other TdT–dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. They demonstrate that TdT–dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10–20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

Read more, please click https://www.nature.com/articles/nbt.4173

3. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos
Rapid, efficient generation of knock-in mice with targeted large insertions remains a major hurdle in mouse genetics. Here, Bin Gu at Hospital for Sick Children in Toronto, Ontario, Canada and his colleagues describe two-cell homologous recombination (2C-HR)-CRISPR, a highly efficient gene-editing method based on introducing CRISPR reagents into embryos at the two-cell stage, which takes advantage of the open chromatin structure and the likely increase in homologous-recombination efficiency during the long G2 phase. Combining 2C-HR-CRISPR with a modified biotin–streptavidin approach to localize repair templates to target sites, they achieved a more-than-tenfold increase (up to 95%) in knock-in efficiency over standard methods. They targeted 20 endogenous genes expressed in blastocysts with fluorescent reporters and generated reporter mouse lines. They also generated triple-color blastocysts with all three lineages differentially labeled, as well as embryos carrying the two-component auxin-inducible degradation system for probing protein function. They suggest that 2C-HR-CRISPR is superior to random transgenesis or standard genome-editing protocols, because it ensures highly efficient insertions at endogenous loci and defined 'safe harbor' sites.

Read more, please click https://www.nature.com/articles/nbt.4166

4. Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER
Precise control over microbial cell growth conditions could enable detection of minute phenotypic changes, which would improve our understanding of how genotypes are shaped by adaptive selection. Although automated cell-culture systems such as bioreactors offer strict control over liquid culture conditions, they often do not scale to high-throughput or require cumbersome redesign to alter growth conditions. Brandon G Wong at Boston University in Boston, Massachusetts, USA and his colleagues report the design and validation of eVOLVER, a scalable do-it-yourself (DIY) framework, which can be configured to carry out high-throughput growth experiments in molecular evolution, systems biology, and microbiology. High-throughput evolution of yeast populations grown at different densities reveals that eVOLVER can be applied to characterize adaptive niches. Growth selection on a genome-wide yeast knockout library, using temperatures varied over different timescales, finds strains sensitive to temperature changes or frequency of temperature change. Inspired by large-scale integration of electronics and microfluidics, they also demonstrate millifluidic multiplexing modules that enable multiplexed media routing, cleaning, vial-to-vial transfers and automated yeast mating.

Read more, please click https://www.nature.com/articles/nbt.4151

5. Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions
Post-translational phosphorylation is essential to human cellular processes, but the transient, heterogeneous nature of this modification complicates its study in native systems. Karl W Barber at Yale University in New Haven, Connecticut, USA and his colleagues developed an approach to interrogate phosphorylation and its role in protein-protein interactions on a proteome-wide scale. They genetically encoded phosphoserine in recoded E. coli and generated a peptide-based heterologous representation of the human serine phosphoproteome. They designed a single-plasmid library encoding >100,000 human phosphopeptides and confirmed the site-specific incorporation of phosphoserine in >36,000 of these peptides. They then integrated their phosphopeptide library into an approach known as Hi-P to enable proteome-level screens for serine-phosphorylation-dependent human protein interactions. Using Hi-P, they found hundreds of known and potentially new phosphoserine-dependent interactors with 14-3-3 proteins and WW domains. These phosphosites retained important binding characteristics of the native human phosphoproteome, as determined by motif analysis and pull-downs using full-length phosphoproteins. This technology can be used to interrogate user-defined phosphoproteomes in any organism, tissue, or disease of interest.

Read more, please click https://www.nature.com/articles/nbt.4150



 

Immunoprecipitation

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or extrinsically labeled cells, subcellular fractions from either unlabeled or labeled cells, or in vitro–translated proteins. Immunoprecipitation is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or sedimentation on density gradients. Either polyclonal or monoclonal antibodies from various animal species can be used in immunoprecipitation protocols. Antibodies can be bound noncovalently to immunoadsorbents such as protein A– or protein G–agarose, or can be coupled covalently to a solid-phase matrix.

Figure 1 Schematic representation of the stages of a typical immunoprecipitation protocol

Immunoprecipitation protocols consist of several stages (Fig 1).
In stage 1, the antigen is solubilized by one of several techniques for lysing cells. Soluble and membrane-associated antigens can be released from cells grown either in suspension culture or as a monolayer on tissue culture dishes with nondenaturing detergents. Cells can also be lysed under denaturing conditions. Soluble antigens can also be extracted by mechanical disruption of cells in the absence of detergents. All of these lysis procedures are suitable for extracting antigens from animal cells. Two commonly used buffers for cell lysis: RIPA buffer gives lower background in IP. However, RIPA can denature some proteins. If you are conducting IP experiments to study protein-protein interactions, RIPA should not be used as it can disrupt the interactions; NP-40 buffer denatures proteins to a lesser extent, and is thus used for phosphorylation experiments when studying kinase activity. NP-40 is typically used for the study of protein-protein interactions. NP-40 is a nonionic detergent and the most commonly used detergent in cell lysis buffers for IP and westerns. Yeast cells require disruption of their cell wall in order to allow extraction of the antigens.

In stage 2, a specific antibody is attached, either noncovalently or covalently, to a sedimentable, solid-phase matrix to allow separation by low-speed centrifugation. For example, the noncovalent attachment of antibody to protein A or protein G agarose beads. Incubation will depend upon the concentration of target protein and the specificity of the Ab toward this target (Table 1).

Table 1 Binding characteristics of different immunoglobulins (Igs).

++, moderate to strong binding; +, weak binding; −, no binding

Stage 3 consists of incubating the solubilized antigen from stage 1 with the immobilized antibody from stage 2, followed by extensive washing to remove unbound proteins. Immunoprecipitated antigens can be dissociated from antibodies and reprecipitated by a protocol referred to as “immunoprecipitation-recapture”. This protocol can be used with the same antibody for further purification of the antigen, or with a second antibody to identify components of multisubunit complexes or to study protein-protein interactions. Immunoprecipitated antigens can be analyzed by one-dimensional electrophoresis, two-dimensional electrophoresis, or immunoblotting. In some cases, immunoprecipitates can be used for structural or functional analyses of the isolated antigens. Immunoprecipitates can also be used as sources of immunogens for production of monoclonal or polyclonal antibodies.

Overview of Cell Fractionation

Cell fractionation has enjoyed widespread use among cell biologists for half a century. It continues to be a fruitful, if not essential, approach in the reductionistic efforts to define the composition and functions of the multiple compartments in eukaryotic cells. It also provides the essential ingredients for the increasing number of cell-free assays now being used in test-tube reconstructions of complex cellular events involving intercompartmental interactions. Much of the knowledge regarding the composition and function of cell organelles has resulted from fractionation of mammalian tissues, where cells are both abundant and highly differentiated (and thus organelle-rich). However, with new techniques in molecular biology and widened interest in combining studies of intact cells and functional reconstitution, interest in fractionation has spread to cultured cell lines and genetically tractable lower eukaryotic cells.

The goals of cell fractionation often differ depending on the nature of the experiments being conducted. In preparative procedures, in which the intent is to isolate quantities of a particular cell organelle for further study or for subfractionation, the emphasis is on purity and (secondarily) on yield. In analytical experiments, in which the intent is not isolation of organelles but evaluation of associations of selected macromolecules with particular organelles, the emphasis is on using one-step procedures that result in different distributions of various organelles (as defined by marker activities) rather than on separating organelles outright. Finally, in preparing organelles for cell-free reconstitution, the goal is to maintain them in a functional state. The investigator generally has developed a specific assay for intercompartmental interaction that does not rely on organelle purity, so the extent of contamination by irrelevant organelles is less important.

The separation of distinct organelles during cell fractionation results from their differing physical properties-size (and shape), buoyant density, and surface charge density-which reflect their differing compositions. Particular fractionation techniques capitalize on one or more of these properties. For example, gel filtration separates on the basis of size, centrifugation separates on the basis of size and density, and electrophoresis separates on the basis of surface charge density. As knowledge of the specific composition of particular organelles has developed, it has become possible to apply newer techniques such as affinity chromatography and selective density-shift perturbation. Whichever fractionation method is used, the procedures may have to be modified to adapt them to individual needs. Even isolation of a particular kind of organelle from different tissue or cell sources may necessitate adjustment of fractionation conditions. This also means that in addition to isolating the organelle of interest, one should also plan on confirming the identity of what has been isolated.

Centrifugation is the most widely used procedure in cell fractionation. It is the only approach commonly used to separate crude tissue homogenates (often having quite large volumes) into subfractions as starting material for more refined purification procedures. Further, the technology available, using rotors with a variety of geometries and diverse media that enable separation according to size, density, or both, now routinely permits refined separations on volumes ranging from submilliliter to several liters. No other technology is this versatile. Gel filtration is limited by the pore sizes of available resins, such that only regularly shaped vesicles with diameters <100 to 200 nm can be purified away from larger or irregularly shaped organelles. Use of electrophoresis for organelle purification, especially at the preparative level, is relatively recent, and, as with gel filtration, its success relies on generating starting material by centrifugation. Although it shows promise for purifying selected organelles (e.g., endosomes) that have been difficult to obtain otherwise, surface charge densities on most organelles may not be different enough (or able to be manipulated sufficiently) to make this a versatile procedure.

Figure 1 The separation of distinct organelles during cell fractionation by centrifugation

Anti-β-Actin Mouse Monoclonal Antibody (1C7), AbFluor™ 488 Conjugated (Cat#A01010A488) Review

β-Actin (gene name ACTB), a ubiquitous eukaryotic protein, is the major component of the cytoskeleton. At least six isoforms are known in mammals. Actins are highly conserved proteins that are involved in cell motility, structure and integrity. β-actin is a major constituent of the contractile apparatus, which is usually used as a loading control, for among others, the integrity of cells, protein degradation, in PCR and Western blotting. Its molecular weight is approximately 43 kDa.

Primary antibody and secondary antibody together to analysis the research results, which not only increased the costs, but extra experimental procedures and time are consumed, may bring more potential of non-specific background. Researchers who want to solve the above problems will choose primary antibodies with direct conjugate.

Abbkine Anti-β-Actin Mouse Monoclonal Antibody (1C7), AbFluor™ 488 Conjugated can apply in IHC (1:200), IF (1:200) without secondary antibody. If your sample is from Chicken, Dog, Hamster, Human, Insect, Monkey, Mouse, Rabbit and Rat, beyond doubt, this antibody is just what you want. And AbFluor™ 488 (λEX/λEm: 490/515 nm) is a green fluorescent dye optimally excitable by the 488 nm argon laser line, which is super alternative to FITC, Oregon Green 488, FAM, Cy2, Dylight 488 and Alexa Fluor 488.



Customer review:
I conduct fluorescent multiple staining. In order to decrease non-specific reaction, I decided to use direct labeled antibody. Abbkine Anti-β-Actin Mouse Monoclonal Antibody (1C7), AbFluor™ 488 Conjugated was recommended by one of our cooperation partner. The antibody was ideal for fluorescence staining. It didn’t let me down. I also really appreciate Abbkine for the wonderful experience it brought to me!

The Nobel Prize in Physiology or Medicine 2012

The Nobel Prize in Physiology or Medicine 2012 was awarded jointly to Sir John B. Gurdon and Shinya Yamanaka "for the discovery that mature cells can be reprogrammed to become pluripotent"

Nobelist	Born	Affiliation at the time of the award
Sir John B. Gurdon	2 October 1933, Dippenhall, United Kingdom	Gurdon Institute, Cambridge, United Kingdom
Shinya Yamanaka	4 September 1962, Osaka, Japan	Kyoto University, Kyoto, Japan, Gladstone Institutes, San Francisco, CA, USA

Summary

The Nobel Prize recognizes two scientists who discovered that mature, specialised cells can be reprogrammed to become immature cells capable of developing into all tissues of the body. Their findings have revolutionised our understanding of how cells and organisms develop.

John B. Gurdon discovered in 1962 that the specialisation of cells is reversible. In a classic experiment, he replaced the immature cell nucleus in an egg cell of a frog with the nucleus from a mature intestinal cell. This modified egg cell developed into a normal tadpole. The DNA of the mature cell still had all the information needed to develop all cells in the frog.

Shinya Yamanaka discovered more than 40 years later, in 2006, how intact mature cells in mice could be reprogrammed to become immature stem cells. Surprisingly, by introducing only a few genes, he could reprogram mature cells to become pluripotent stem cells, i.e. immature cells that are able to develop into all types of cells in the body.

These groundbreaking discoveries have completely changed our view of the development and cellular specialisation. We now understand that the mature cell does not have to be confined forever to its specialised state. Textbooks have been rewritten and new research fields have been established. By reprogramming human cells, scientists have created new opportunities to study diseases and develop methods for diagnosis and therapy.

More details, please click The 2012 Nobel Prize in Physiology or Medicine.

The Nobel Prize in Physiology or Medicine 2012

The Nobel Prize in Physiology or Medicine 2012 was awarded jointly to Sir John B. Gurdon and Shinya Yamanaka "for the discovery that mature cells can be reprogrammed to become pluripotent"

Nobelist	Born	Affiliation at the time of the award
Sir John B. Gurdon	2 October 1933, Dippenhall, United Kingdom	Gurdon Institute, Cambridge, United Kingdom
Shinya Yamanaka	4 September 1962, Osaka, Japan	Kyoto University, Kyoto, Japan, Gladstone Institutes, San Francisco, CA, USA

Summary

The Nobel Prize recognizes two scientists who discovered that mature, specialised cells can be reprogrammed to become immature cells capable of developing into all tissues of the body. Their findings have revolutionised our understanding of how cells and organisms develop.

John B. Gurdon discovered in 1962 that the specialisation of cells is reversible. In a classic experiment, he replaced the immature cell nucleus in an egg cell of a frog with the nucleus from a mature intestinal cell. This modified egg cell developed into a normal tadpole. The DNA of the mature cell still had all the information needed to develop all cells in the frog.

Shinya Yamanaka discovered more than 40 years later, in 2006, how intact mature cells in mice could be reprogrammed to become immature stem cells. Surprisingly, by introducing only a few genes, he could reprogram mature cells to become pluripotent stem cells, i.e. immature cells that are able to develop into all types of cells in the body.

These groundbreaking discoveries have completely changed our view of the development and cellular specialisation. We now understand that the mature cell does not have to be confined forever to its specialised state. Textbooks have been rewritten and new research fields have been established. By reprogramming human cells, scientists have created new opportunities to study diseases and develop methods for diagnosis and therapy.

More details, please click The 2012 Nobel Prize in Physiology or Medicine.

Regulation of thymocyte trafficking by Tagap, a GAP domain protein linked to human autoimmunity

Content introduction:

• A live vaccine rapidly protects against cholera in an infant rabbit model


• NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension


• Regulation of thymocyte trafficking by Tagap, a GAP domain protein linked to human autoimmunity


• “Disruptor” residues in the regulator of G protein signaling (RGS) R12 subfamily attenuate the inactivation of Gα subunits


• Statins enhance efficacy of venetoclax in blood cancers

1. A live vaccine rapidly protects against cholera in an infant rabbit model
Outbreaks of cholera, a rapidly fatal diarrheal disease, often spread explosively. The efficacy of reactive vaccination campaigns—deploying Vibrio cholerae vaccines during epidemics—is partially limited by the time required for vaccine recipients to develop adaptive immunity. Troy P. Hubbard at Harvard Medical School in Boston, USA and his colleagues created HaitiV, a live attenuated cholera vaccine candidate, by deleting diarrheagenic factors from a recent clinical isolate of V. cholerae and incorporating safeguards against vaccine reversion. They demonstrate that administration of HaitiV 24 hours before lethal challenge with wild-type V. cholerae reduced intestinal colonization by the wild-type strain, slowed disease progression, and reduced mortality in an infant rabbit model of cholera. HaitiV-mediated protection required viable vaccine, and rapid protection kinetics are not consistent with development of adaptive immunity. These features suggest that HaitiV mediates probiotic-like protection from cholera, a mechanism that is not known to be elicited by traditional vaccines. Mathematical modeling indicates that an intervention that works at the speed of HaitiV-mediated protection could improve the public health impact of reactive vaccination.

Read more, please click http://stm.sciencemag.org/content/10/445/eaap8423

2. NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension
Germline mutations involving small mothers against decapentaplegic–transforming growth factor–β (SMAD–TGF-β) signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is a disease characterized, in part, by vascular fibrosis and hyperaldosteronism (ALDO). Andriy O. Samokhin at Brigham and Women’s Hospital in Boston, USA and his colleagues developed and analyzed a fibrosis protein-protein network (fibrosome) in silico, which predicted that the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical ALDO-regulated node underpinning pathogenic vascular fibrosis. Bioinformatics and microscale thermophoresis demonstrated that oxidation of Cys18 in the SMAD3 docking region of NEDD9 impairs SMAD3-NEDD9 protein-protein interactions in vitro. This effect was reproduced by ALDO-induced oxidant stress in cultured human pulmonary artery endothelial cells (HPAECs), resulting in impaired NEDD9 proteolytic degradation, increased NEDD9 complex formation with Nk2 homeobox 5 (NKX2-5), and increased NKX2-5 binding to COL3A1. Up-regulation of NEDD9-dependent collagen III expression corresponded to changes in cell stiffness measured by atomic force microscopy. HPAEC-derived exosomal signaling targeted NEDD9 to increase collagen I/III expression in human pulmonary artery smooth muscle cells, identifying a second endothelial mechanism regulating vascular fibrosis. ALDO-NEDD9 signaling was not affected by treatment with a TGF-β ligand trap and, thus, was not contingent on TGF-β signaling. Colocalization of NEDD9 with collagen III in HPAECs was observed in fibrotic pulmonary arterioles from PAH patients. Furthermore, NEDD9 ablation or inhibition prevented fibrotic vascular remodeling and pulmonary hypertension in animal models of PAH in vivo. These data identify a critical TGF-β–independent posttranslational modification that impairs SMAD3-NEDD9 binding in HPAECs to modulate vascular fibrosis and promote PAH.

Read more, please click http://stm.sciencemag.org/content/10/445/eaap7294

3. Regulation of thymocyte trafficking by Tagap, a GAP domain protein linked to human autoimmunity
Multiple autoimmune pathologies are associated with single-nucleotide polymorphisms of the human gene TAGAP, which encodes TAGAP, a guanosine triphosphatase (GTPase)–activating protein. Jonathan S. Duke-Cohan at Dana-Farber Cancer Institute in Boston, USA and his colleagues showed in mice that Tagap-mediated signaling by the sema3E/plexin-D1 ligand-receptor complex attenuates thymocytes’ adhesion to the cortex through their β1-containing integrins. By promoting thymocyte detachment within the cortex of the thymus, Tagap-mediated signaling enabled their translocation to the medulla, which is required for continued thymic selection. Tagap physically interacted with the cytoplasmic domain of plexin-D1 and directly stimulated the activity and signaling of the GTPase RhoA. In addition, Tagap indirectly mediated the activation of Cdc42 in response to the binding of sema3E to plexin-D1. Both RhoA and Cdc42 are key mediators of cytoskeletal and integrin dynamics in thymocytes. Knockdown of Tagap in mice suppressed the sema3E- and plexin-D1–mediated release of thymocytes that adhered within the cortex through β1-containing integrins. This suppression led to the impaired translocation of thymocytes from the cortex to the medulla and resulted in the formation of ectopic medullary structures within the thymic cortex. Their results suggest that TAGAP variation modulates the risk of autoimmunity by altering thymocyte migration during thymic selection.



Read more, please click http://stke.sciencemag.org/content/11/534/eaan8799

4. “Disruptor” residues in the regulator of G protein signaling (RGS) R12 subfamily attenuate the inactivation of Gα subunits
Understanding the molecular basis of interaction specificity between RGS (regulator of G protein signaling) proteins and heterotrimeric (αβγ) G proteins would enable the manipulation of RGS-G protein interactions, explore their functions, and effectively target them therapeutically. RGS proteins are classified into four subfamilies (R4, R7, RZ, and R12) and function as negative regulators of G protein signaling by inactivating Gα subunits. Ali Asli at University of Haifa in Haifa, Israel and her colleagues found that the R12 subfamily members RGS10 and RGS14 had lower activity than most R4 subfamily members toward the Gi subfamily member Gαo. Using structure-based energy calculations with multiple Gα-RGS complexes, they identified R12-specific residues in positions that are predicted to determine the divergent activity of this subfamily. This analysis predicted that these residues, which they call “disruptor residues,” interact with the Gα helical domain. They engineered the R12 disruptor residues into the RGS domains of the high-activity R4 subfamily and found that these altered proteins exhibited reduced activity toward Gαo. Reciprocally, replacing the putative disruptor residues in RGS18 (a member of the R4 subfamily that exhibited low activity toward Gαo) with the corresponding residues from a high-activity R4 subfamily RGS protein increased its activity toward Gαo. Furthermore, the high activity of the R4 subfamily toward Gαo was independent of the residues in the homologous positions to the R12 subfamily and RGS18 disruptor residues. Thus, their results suggest that the identified RGS disruptor residues function as negative design elements that attenuate RGS activity for specific Gα proteins.

Read more, please click http://stke.sciencemag.org/content/11/534/eaan3677

5. Statins enhance efficacy of venetoclax in blood cancers
Statins have shown promise as anticancer agents in experimental and epidemiologic research. However, any benefit that they provide is likely context-dependent, for example, applicable only to certain cancers or in combination with specific anticancer drugs. J. Scott Lee at University of California in Irvine, USA and his colleagues report that inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) using statins enhances the proapoptotic activity of the B cell lymphoma-2 (BCL2) inhibitor venetoclax (ABT-199) in primary leukemia and lymphoma cells but not in normal human peripheral blood mononuclear cells. By blocking mevalonate production, HMGCR inhibition suppressed protein geranylgeranylation, resulting in up-regulation of proapoptotic protein p53 up-regulated modulator of apoptosis (PUMA). In support of these findings, dynamic BH3 profiling confirmed that statins primed cells for apoptosis. Furthermore, in retrospective analyses of three clinical studies of chronic lymphocytic leukemia, background statin use was associated with enhanced response to venetoclax, as demonstrated by more frequent complete responses. Together, this work provides mechanistic justification and clinical evidence to warrant prospective clinical investigation of this combination in hematologic malignancies.

Read more, please click http://stm.sciencemag.org/content/10/445/eaaq1240

Overview of the Physical State of Proteins Within Cells

The word protein comes from the Greek word proteios, meaning primary. And, indeed, proteins are of primary importance in the study of cell function. It is difficult to imagine a cellular function not linked with proteins. Almost all biochemical catalysis is carried out by protein enzymes. Proteins participate in gene regulation, transcription, and translation. Intracellular filaments give shape to a cell while extracellular proteins hold cells together to form organs. Proteins transport other molecules, such as oxygen, to tissues. Antibody molecules contribute to host defense against infections. Protein hormones relay information between cells. Moreover, protein machines, such as actin-myosin complexes, can perform useful work including cell movement. Thus, studying proteins is a prerequisite in understanding cell structure and function.

The physical characterization of proteins began well over 150 years ago with Mulder’s characterization of the atomic composition of proteins. In the latter half of the nineteenth century Hoppe-Seyler (1864) crystallized hemoglobin and Kühn (1876) purified trypsin. A variety of physical methods have been developed over the years to increase convenience and precision in the characterization and isolation of proteins. These include ultracentrifugation, chromatography, electrophoresis, and others. In many instances our understanding of cell proteins parallels the introduction and use of new techniques to examine their structure and function.

All proteins are constructed as a linear sequence(s) of various numbers and combinations of ∼20 α-amino acids joined by peptide bonds to form structures from thousands to millions of daltons in size. Proteins are the most complex and heterogeneous molecules found in cells, where they account for >50% of the dry weight of cells and ∼75% of tissues. Proteins can be classified into three broad groups: globular, fibrous, and transmembrane (Table 1, Figure 1). Globular proteins are, by definition, globe-shaped, although in practice they can be spherical or ellipsoidal. Globular proteins are generally soluble in aqueous environments. Examples of globular proteins are hemoglobin, serum albumin, and most enzymes. Fibrous proteins are elongated linear molecules that are generally insoluble in water and resist applied stresses and strains. Collagen is a physically tough molecule of connective tissue. Just as collagen gives strength to connective tissues, intermediate filaments linked to desmosomes give strength to cells in tissues. The third general class of proteins, transmembrane proteins, contain a hydrophobic sequence buried within the membrane. These protein categories are not mutually exclusive. For example, the nominally fibrous intermediate filament proteins also have globular domains. Similarly, transmembrane proteins almost always possess globular domains. Thus, these definitions serve as a useful guide but should not be rigidly applied.

Figure 1 General classifications of proteins.

In these schematic representations of globular, fibrous, and transmembrane proteins, hydrophobic regions are shaded. Note that the disposition of hydrophobic residues often reflects the protein class.

Table 1 Broad Classifications for Proteins

A key physical feature of proteins is their hydropathy pattern (i.e., the distribution of hydrophobic and hydrophilic amino acid residues). Indeed, hydrophobic interactions provide the primary net free energy required for protein folding. Figure 1 illustrates the disposition of hydrophobic amino acids in proteins. In an intact globular protein, hydrophobic amino acids are generally shielded from the aqueous environment by coalescing at the center of the molecule, with the more hydrophilic residues exposed at its surface. However, the linear arrangement of hydrophobic residues fluctuates in an apparently random fashion. The α helices within globular proteins may express a hydrophobic face oriented toward the center of the protein. Within these helices hydrophobic residues are nonrandomly positioned every three or four amino acids to yield a hydrophobic face. For coiled-coil α helix–containing fibrous proteins, such as tropomyosin and α-keratin, hydrophobic residues at periodic intervals allow close van der Waals contact of the chains and potentiate assembly as hydrophobic residues are removed from the aqueous environment. Secondarily, regularly spaced charged groups can also contribute to the shape of fibrous proteins. Transmembrane proteins provide a rather different physical arrangement of hydrophobic residues in which hydrophobic residues are collected primarily into a series of amino acids that is embedded within a cell membrane. One important means of analyzing the hydropathy of a sequenced protein is a hydropathy plot. In this method, each amino acid residue is assigned a hydropathy value, an ad hoc measure that largely reflects its relative aqueous solubility; these values are plotted after being averaged. The successful interpretation of hydropathy plots depends on the parameters chosen for averaging. The parameters are the number of residues averaged (amino acid interval or “window”) and how many amino acids are skipped when calculating the next average (step size). Using this approach with a window of ∼10 residues, it is often possible to find the positions of hydrophobic residues coalescing near the interior of globular proteins. The method is particularly useful in predicting transmembrane domains of proteins, generally with a window of ∼20 amino acids. To detect the repetitious pattern of coiled-coil fibrous proteins, however, windows smaller than the repeat length would be required.

Overview of the Physical State of Proteins Within Cells

The word protein comes from the Greek word proteios, meaning primary. And, indeed, proteins are of primary importance in the study of cell function. It is difficult to imagine a cellular function not linked with proteins. Almost all biochemical catalysis is carried out by protein enzymes. Proteins participate in gene regulation, transcription, and translation. Intracellular filaments give shape to a cell while extracellular proteins hold cells together to form organs. Proteins transport other molecules, such as oxygen, to tissues. Antibody molecules contribute to host defense against infections. Protein hormones relay information between cells. Moreover, protein machines, such as actin-myosin complexes, can perform useful work including cell movement. Thus, studying proteins is a prerequisite in understanding cell structure and function.

The physical characterization of proteins began well over 150 years ago with Mulder’s characterization of the atomic composition of proteins. In the latter half of the nineteenth century Hoppe-Seyler (1864) crystallized hemoglobin and Kühn (1876) purified trypsin. A variety of physical methods have been developed over the years to increase convenience and precision in the characterization and isolation of proteins. These include ultracentrifugation, chromatography, electrophoresis, and others. In many instances our understanding of cell proteins parallels the introduction and use of new techniques to examine their structure and function.

All proteins are constructed as a linear sequence(s) of various numbers and combinations of ∼20 α-amino acids joined by peptide bonds to form structures from thousands to millions of daltons in size. Proteins are the most complex and heterogeneous molecules found in cells, where they account for >50% of the dry weight of cells and ∼75% of tissues. Proteins can be classified into three broad groups: globular, fibrous, and transmembrane (Table 1, Figure 1). Globular proteins are, by definition, globe-shaped, although in practice they can be spherical or ellipsoidal. Globular proteins are generally soluble in aqueous environments. Examples of globular proteins are hemoglobin, serum albumin, and most enzymes. Fibrous proteins are elongated linear molecules that are generally insoluble in water and resist applied stresses and strains. Collagen is a physically tough molecule of connective tissue. Just as collagen gives strength to connective tissues, intermediate filaments linked to desmosomes give strength to cells in tissues. The third general class of proteins, transmembrane proteins, contain a hydrophobic sequence buried within the membrane. These protein categories are not mutually exclusive. For example, the nominally fibrous intermediate filament proteins also have globular domains. Similarly, transmembrane proteins almost always possess globular domains. Thus, these definitions serve as a useful guide but should not be rigidly applied.

Figure 1 General classifications of proteins.

In these schematic representations of globular, fibrous, and transmembrane proteins, hydrophobic regions are shaded. Note that the disposition of hydrophobic residues often reflects the protein class.

Table 1 Broad Classifications for Proteins

A key physical feature of proteins is their hydropathy pattern (i.e., the distribution of hydrophobic and hydrophilic amino acid residues). Indeed, hydrophobic interactions provide the primary net free energy required for protein folding. Figure 1 illustrates the disposition of hydrophobic amino acids in proteins. In an intact globular protein, hydrophobic amino acids are generally shielded from the aqueous environment by coalescing at the center of the molecule, with the more hydrophilic residues exposed at its surface. However, the linear arrangement of hydrophobic residues fluctuates in an apparently random fashion. The α helices within globular proteins may express a hydrophobic face oriented toward the center of the protein. Within these helices hydrophobic residues are nonrandomly positioned every three or four amino acids to yield a hydrophobic face. For coiled-coil α helix–containing fibrous proteins, such as tropomyosin and α-keratin, hydrophobic residues at periodic intervals allow close van der Waals contact of the chains and potentiate assembly as hydrophobic residues are removed from the aqueous environment. Secondarily, regularly spaced charged groups can also contribute to the shape of fibrous proteins. Transmembrane proteins provide a rather different physical arrangement of hydrophobic residues in which hydrophobic residues are collected primarily into a series of amino acids that is embedded within a cell membrane. One important means of analyzing the hydropathy of a sequenced protein is a hydropathy plot. In this method, each amino acid residue is assigned a hydropathy value, an ad hoc measure that largely reflects its relative aqueous solubility; these values are plotted after being averaged. The successful interpretation of hydropathy plots depends on the parameters chosen for averaging. The parameters are the number of residues averaged (amino acid interval or “window”) and how many amino acids are skipped when calculating the next average (step size). Using this approach with a window of ∼10 residues, it is often possible to find the positions of hydrophobic residues coalescing near the interior of globular proteins. The method is particularly useful in predicting transmembrane domains of proteins, generally with a window of ∼20 amino acids. To detect the repetitious pattern of coiled-coil fibrous proteins, however, windows smaller than the repeat length would be required.

Fasting Activates Fatty Acid Oxidation to Enhance Intestinal Stem Cell Function during Homeostasis and Aging

Content introduction:

• Genomic Features of Response to Combination Immunotherapy in Patients with Advanced Non-Small-Cell Lung Cancer


• Tumor Mutational Burden and Efficacy of Nivolumab Monotherapy and in Combination with Ipilimumab in Small-Cell Lung Cancer


• A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment


• Prolonged Fasting Reduces IGF-1/PKA to Promote Hematopoietic-Stem-Cell-Based Regeneration and Reverse Immunosuppression


• Fasting Activates Fatty Acid Oxidation to Enhance Intestinal Stem Cell Function during Homeostasis and Aging

1. Genomic Features of Response to Combination Immunotherapy in Patients with Advanced Non-Small-Cell Lung Cancer
Combination immune checkpoint blockade has demonstrated promising benefit in lung cancer, but predictors of response to combination therapy are unknown. Using whole-exome sequencing to examine non-small-cell lung cancer (NSCLC) treated with PD-1 plus CTLA-4 blockade, Matthew D. Hellmann at Memorial Sloan Kettering Cancer Center in New York, USA and his colleagues found that high tumor mutation burden (TMB) predicted improved objective response, durable benefit, and progression-free survival. TMB was independent of PD-L1 expression and the strongest feature associated with efficacy in multivariable analysis. The low response rate in TMB low NSCLCs demonstrates that combination immunotherapy does not overcome the negative predictive impact of low TMB. This study demonstrates the association between TMB and benefit to combination immunotherapy in NSCLC. TMB should be incorporated in future trials examining PD-(L)1 with CTLA-4 blockade in NSCLC.

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30123-5

2. Tumor Mutational Burden and Efficacy of Nivolumab Monotherapy and in Combination with Ipilimumab in Small-Cell Lung Cancer
Durable responses and encouraging survival have been demonstrated with immune checkpoint inhibitors in small-cell lung cancer (SCLC), but predictive markers are unknown. Matthew D. Hellmann at Weill Cornell Medical College, and Parker Center for Cancer Immunotherapy in New York, USA and his colleagues used whole exome sequencing to evaluate the impact of tumor mutational burden on efficacy of nivolumab monotherapy or combined with ipilimumab in patients with SCLC from the nonrandomized or randomized cohorts of CheckMate 032. Patients received nivolumab (3 mg/kg every 2 weeks) or nivolumab plus ipilimumab (1 mg/kg plus 3 mg/kg every 3 weeks for four cycles, followed by nivolumab 3 mg/kg every 2 weeks). Efficacy of nivolumab ± ipilimumab was enhanced in patients with high tumor mutational burden. Nivolumab plus ipilimumab appeared to provide a greater clinical benefit than nivolumab monotherapy in the high tumor mutational burden tertile.

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30172-7

3. A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment
Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. Amelie Griveau at University of California San Francisco in San Francisco, USA and his colleagues show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30125-9

4. Prolonged Fasting Reduces IGF-1/PKA to Promote Hematopoietic-Stem-Cell-Based Regeneration and Reverse Immunosuppression
Immune system defects are at the center of aging and a range of diseases. Here, Chia-Wei Cheng at University of Southern California in Los Angeles, USA and his colleagues show that prolonged fasting reduces circulating IGF-1 levels and PKA activity in various cell populations, leading to signal transduction changes in long-term hematopoietic stem cells (LT-HSCs) and niche cells that promote stress resistance, self-renewal, and lineage-balanced regeneration. Multiple cycles of fasting abated the immunosuppression and mortality caused by chemotherapy and reversed age-dependent myeloid-bias in mice, in agreement with preliminary data on the protection of lymphocytes from chemotoxicity in fasting patients. The proregenerative effects of fasting on stem cells were recapitulated by deficiencies in either IGF-1 or PKA and blunted by exogenous IGF-1. These findings link the reduced levels of IGF-1 caused by fasting to PKA signaling and establish their crucial role in regulating hematopoietic stem cell protection, self-renewal, and regeneration.

Read more, please click https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(14)00151-9

5. Fasting Activates Fatty Acid Oxidation to Enhance Intestinal Stem Cell Function during Homeostasis and Aging
Diet has a profound effect on tissue regeneration in diverse organisms, and low caloric states such as intermittent fasting have beneficial effects on organismal health and age-associated loss of tissue function. The role of adult stem and progenitor cells in responding to short-term fasting and whether such responses improve regeneration are not well studied. Here Maria M. Mihaylova at Whitehead Institute for Biomedical Research in Cambridge, USA and her colleagues show that a 24 hr fast augments intestinal stem cell (ISC) function in young and aged mice by inducing a fatty acid oxidation (FAO) program and that pharmacological activation of this program mimics many effects of fasting. Acute genetic disruption of Cpt1a, the rate-limiting enzyme in FAO, abrogates ISC-enhancing effects of fasting, but long-term Cpt1a deletion decreases ISC numbers and function, implicating a role for FAO in ISC maintenance. These findings highlight a role for FAO in mediating pro-regenerative effects of fasting in intestinal biology, and they may represent a viable strategy for enhancing intestinal regeneration.



Read more, please click https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(18)30163-2

The Nobel Prize in Physiology or Medicine 2013

The Nobel Prize in Physiology or Medicine 2013 was awarded jointly to James E. Rothman, Randy W. Schekman and Thomas C. Südhof "for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells".

Nobelist	Born	Affiliation at the time of the award
James E. Rothman	3 November 1950, Haverhill, MA, USA	Yale University, New Haven, CT, USA
Randy W. Schekman	30 December 1948, St. Paul, MN, USA	University of California, Berkeley, CA, USA, Howard Hughes Medical Institute
Thomas C. Südhof	22 December 1955, Göttingen, Germany	Stanford University, Stanford, CA, USA, Howard Hughes Medical Institute

Summary

The 2013 Nobel Prize honours three scientists who have solved the mystery of how the cell organizes its transport system. Each cell is a factory that produces and exports molecules. For instance, insulin is manufactured and released into the blood and signaling molecules called neurotransmitters are sent from one nerve cell to another. These molecules are transported around the cell in small packages called vesicles. The three Nobel Laureates have discovered the molecular principles that govern how this cargo is delivered to the right place at the right time in the cell.

Randy Schekman discovered a set of genes that were required for vesicle traffic. James Rothman  unravelled protein machinery that allows vesicles to fuse with their targets to permit transfer of cargo. Thomas Südhof revealed how signals instruct vesicles to release their cargo with precision.

Through their discoveries, Rothman, Schekman and Südhof have revealed the exquisitely precise control system for the transport and delivery of cellular cargo. Disturbances in this system have deleterious effects and contribute to conditions such as neurological diseases, diabetes, and immunological disorders.

More details, please click The 2013 Nobel Prize in Physiology or Medicine.

The Nobel Prize in Physiology or Medicine 2013

The Nobel Prize in Physiology or Medicine 2013 was awarded jointly to James E. Rothman, Randy W. Schekman and Thomas C. Südhof "for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells".

Nobelist	Born	Affiliation at the time of the award
James E. Rothman	3 November 1950, Haverhill, MA, USA	Yale University, New Haven, CT, USA
Randy W. Schekman	30 December 1948, St. Paul, MN, USA	University of California, Berkeley, CA, USA, Howard Hughes Medical Institute
Thomas C. Südhof	22 December 1955, Göttingen, Germany	Stanford University, Stanford, CA, USA, Howard Hughes Medical Institute

Summary

The 2013 Nobel Prize honours three scientists who have solved the mystery of how the cell organizes its transport system. Each cell is a factory that produces and exports molecules. For instance, insulin is manufactured and released into the blood and signaling molecules called neurotransmitters are sent from one nerve cell to another. These molecules are transported around the cell in small packages called vesicles. The three Nobel Laureates have discovered the molecular principles that govern how this cargo is delivered to the right place at the right time in the cell.

Randy Schekman discovered a set of genes that were required for vesicle traffic. James Rothman  unravelled protein machinery that allows vesicles to fuse with their targets to permit transfer of cargo. Thomas Südhof revealed how signals instruct vesicles to release their cargo with precision.

Through their discoveries, Rothman, Schekman and Südhof have revealed the exquisitely precise control system for the transport and delivery of cellular cargo. Disturbances in this system have deleterious effects and contribute to conditions such as neurological diseases, diabetes, and immunological disorders.

More details, please click The 2013 Nobel Prize in Physiology or Medicine.

The Nobel Prize in Physiology or Medicine 2014

The Nobel Prize in Physiology or Medicine 2014 was divided, one half awarded to John O'Keefe, the other half jointly to May-Britt Moser and Edvard I. Moser "for their discoveries of cells that constitute a positioning system in the brain".

Nobelist	Born	Affiliation at the time of the award
John O'Keefe	18 November 1939, New York, NY, USA	University College, London, United Kingdom
May-Britt Moser	4 January 1963, Fosnavåg, Norway	Norwegian University of Science and Technology (NTNU), Trondheim, Norway
Edvard I. Moser	27 April 1962, Ålesund, Norway	Norwegian University of Science and Technology (NTNU), Trondheim, Norway

How do we know where we are? How can we find the way from one place to another? And how can we store this information in such a way that we can immediately find the way the next time we trace the same path? The 2014 year's Nobel Laureates have discovered a positioning system, an "inner GPS" in the brain that makes it possible to orient ourselves in space, demonstrating a cellular basis for higher cognitive function.

More details, please click The 2014 Nobel Prize in Physiology or Medicine.

Structural basis of ubiquitin modification by the Legionella effector SdeA

Content introduction:

• Pyramidal cell regulation of interneuron survival sculpts cortical networks


• Cortical direction selectivity emerges at convergence of thalamic synapses


• Structural basis of ubiquitin modification by the Legionella effector SdeA


• ANKRD16 prevents neuron loss caused by an editing-defective tRNA synthetase


• Structure of a volume-regulated anion channel of the LRRC8 family

1. Pyramidal cell regulation of interneuron survival sculpts cortical networks
Complex neuronal circuitries such as those found in the mammalian cerebral cortex have evolved as balanced networks of excitatory and inhibitory neurons. Although the establishment of appropriate numbers of these cells is essential for brain function and behaviour, our understanding of this fundamental process is limited. Here Fong Kuan Wong at King’s College London in London, UK and his colleagues show that the survival of interneurons in mice depends on the activity of pyramidal cells in a critical window of postnatal development, during which excitatory synaptic input to individual interneurons predicts their survival or death. Pyramidal cells regulate interneuron survival through the negative modulation of PTEN signalling, which effectively drives interneuron cell death during this period. Their findings indicate that activity-dependent mechanisms dynamically adjust the number of inhibitory cells in nascent local cortical circuits, ultimately establishing the appropriate proportions of excitatory and inhibitory neurons in the cerebral cortex.

Read more, please click https://www.nature.com/articles/s41586-018-0139-6

2. Cortical direction selectivity emerges at convergence of thalamic synapses
Detecting the direction of motion of an object is essential for our representation of the visual environment. The visual cortex is one of the main stages in the mammalian nervous system in which the direction of motion may be computed de novo. Experiments and theories indicate that cortical neurons respond selectively to motion direction by combining inputs that provide information about distinct spatial locations with distinct time delays. Despite the importance of this spatiotemporal offset for direction selectivity, its origin and cellular mechanisms are not fully understood. Anthony D. Lien at University of California San Diego in La Jolla, USA and his colleagues show that approximately 80 ± 10 thalamic neurons, which respond with distinct time courses to stimuli in distinct locations, excite mouse visual cortical neurons during visual stimulation. The integration of thalamic inputs with the appropriate spatiotemporal offset provides cortical neurons with a primordial bias for direction selectivity. These data show how cortical neurons selectively combine the spatiotemporal response diversity of thalamic neurons to extract fundamental features of the visual world.

Read more, please click https://www.nature.com/articles/s41586-018-0148-5

3. Structural basis of ubiquitin modification by the Legionella effector SdeA
Protein ubiquitination is a multifaceted post-translational modification that controls almost every process in eukaryotic cells. Recently, the Legionella effector SdeA was reported to mediate a unique phosphoribosyl-linked ubiquitination through successive modifications of the Arg42 of ubiquitin (Ub) by its mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains. However, the mechanisms of SdeA-mediated Ub modification and phosphoribosyl-linked ubiquitination remain unknown. Here Yanan Dong at Beijing University of Chemical Technology in Beijing, China and his colleagues report the structures of SdeA in its ligand-free, Ub-bound and Ub–NADH-bound states. The structures reveal that the mART and PDE domains of SdeA form a catalytic domain over its C-terminal region. Upon Ub binding, the canonical ADP-ribosyltransferase toxin turn-turn (ARTT) and phosphate-nicotinamide (PN) loops in the mART domain of SdeA undergo marked conformational changes. The Ub Arg72 might act as a ‘probe’ that interacts with the mART domain first, and then movements may occur in the side chains of Arg72 and Arg42 during the ADP-ribosylation of Ub. Their study reveals the mechanism of SdeA-mediated Ub modification and provides a framework for further investigations into the phosphoribosyl-linked ubiquitination process.



Read more, please click https://www.nature.com/articles/s41586-018-0146-7

4. ANKRD16 prevents neuron loss caused by an editing-defective tRNA synthetase
Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aarssti mutant mice results in an increase in the production of serine-mischarged tRNAAla and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, My-Nuong Vo at Scripps Research Institute in La Jolla, USA and his colleagues identified Ankrd16, a gene that acts epistatically with the Aarssti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNAAla and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aarssti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.

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5. Structure of a volume-regulated anion channel of the LRRC8 family
Volume-regulated anion channels are activated in response to hypotonic stress. These channels are composed of closely related paralogues of the leucine-rich repeat-containing protein 8 (LRRC8) family that co-assemble to form hexameric complexes. Here, using cryo-electron microscopy and X-ray crystallography, Dawid Deneka at University of Zurich in Zurich, Switzerland and his colleagues determine the structure of a homomeric channel of the obligatory subunit LRRC8A. This protein conducts ions and has properties in common with endogenous heteromeric channels. Its modular structure consists of a transmembrane pore domain followed by a cytoplasmic leucine-rich repeat domain. The transmembrane domain, which is structurally related to connexin proteins, is wide towards the cytoplasm but constricted on the outside by a structural unit that acts as a selectivity filter. An excess of basic residues in the filter and throughout the pore attracts anions by electrostatic interaction. Their work reveals the previously unknown architecture of volume-regulated anion channels and their mechanism of selective anion conduction.

Read more, please click https://www.nature.com/articles/s41586-018-0134-y

Antibodies as Cell Biological Tools

Monoclonal and polyclonal antibodies are powerful tools for addressing cell biological questions. These immunological reagents can be used to detect and analyze proteins and carbohydrates, characterize the subcellular distribution of proteins, and purify proteins. In addition, when taken up by cells, antibodies can be used as tags to monitor the intracellular pathways of proteins and to inhibit protein activity. These diverse applications of antibody technology have been facilitated by advances in the understanding of the molecular genetics of antibody molecules and their three-dimensional structures. The development of methods for measuring antibody binding activity and for isolating antibodies has contributed to their widespread use and extensive range of applications.

The development of monoclonal antibodies, unique and powerful reagents for detecting and measuring interactions with specific protein epitopes, has revolutionized the use of antibodies in cell biological research. These antibodies are obtained by fusing immune B cells from the spleen with tumor cells to produce hybridomas. Hybridomas each secrete a single type of antibody—the monoclonal antibody—that has precise specificity and often high affinity. Because the cloned hybridoma cell line is immortal, with appropriate care it can be maintained indefinitely and the antibodies it produces can be supplied in essentially limitless quantities. Preparations containing monoclonal antibodies include hybridoma supernatants, ascites fluid from a mouse inoculated with the hybridoma, and purified monoclonal antibody.

Polyclonal antibodies take less effort to prepare than monoclonal antibodies. The process consists simply of immunizing an animal of any one of a variety of species (including goat, horse, rat, mouse, and rabbit) with purified antigen and then, after the animal develops an immune response, isolating antibodies from its serum. Because polyclonal antibodies are essentially a collection of monoclonal antibodies with different epitope specificities and affinities, they are useful for analyses of denatured forms of a protein, for immunoprecipitation and immunoblotting. These types of analyses are often not successful with monoclonal antibodies because the single epitope recognized by the monoclonal antibody preparation may be destroyed during protein denaturation.

The choice of animal species for immunization depends in part on the amount of antiserum required for subsequent experiments. Mice, rats, and guinea pigs yield relatively low volumes of antiserum compared to rabbits and other larger animals. For this reason, rabbits are often the animals of choice. It is often desirable to produce polyclonal antibodies in other species, however, especially when an experiment requires two distinct types of antibodies that recognize different proteins (as is often the case in indirect immunofluorescence assays).



The term "antibody production" has both general and specific meanings. In the broad sense, it refers to the entire process of creating a usable specific antibody, including steps of immunogen preparation, immunization, hybridoma creation, collection, screening, isotyping, purification, and labeling for direct use in a particular method. In the more restricted sense, antibody production refers to the steps leading up to antibody generation but does not include various forms of purifying and labeling the antibody for particular uses.Successful antibody production depends upon careful planning and implementation with respect to several important steps and considerations:
1. Synthesizing or purifying the target antigen (e.g., peptide or hapten)；
2. Choosing an appropriate immunogenic carrier protein；
3. Conjugating the antigen and carrier protein to create the immunogen；
4. Immunizing animals using appropriate schedules and adjuvant formulae；
5. Screening serum (or hybridomas) for antibody titer and isotype；
Procedures for generating, purifying and modifying antibodies for use as antigen-specific probes were developed during the 1970s and 1980s and have remained relatively unchanged since Harlow and Lane published their classic Antibodies: A Laboratory Manual in 1988.