Horseradish peroxidase (HRP) is a 44,173.9-dalton glycoprotein with 6 lysine residues which can be conjugated to a labeled molecule. It produces a colored, fluorometric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified. HRP is often used in conjugates to determine the presence of a molecular target. HRP is also commonly used in techniques such as ELISA and Immunohistochemistry due to its monomeric nature and the ease with which it produces colored products.
Abbkine LinKine™ HRP Labeling Kit is designed for preparing HRP conjugates directly from proteins, peptides, and other ligands that contain a free amino group. The HRP provided in our kit has been pre-activated and can be directly used for conjugation. LinKine™ HRP conjugate Kit contains ready-to-use components to prepare HRP labeled molecules. The resulting conjugates are stable for at least 6 months at 4˚C.
Why should you pick Abbkine LinKine™ HRP Labeling Kit?
Activated HRP – periodate-treated, aldehyde-activated horseradish peroxidase, ready for Labeling to antibodies and other proteins.
High activity HRP – enzyme activity is 120 to 200 units/mg; activated enzyme is stable for at least 12 months at -20°C.
Convenient quantities – each 1 mg-quantity of activated enzyme is sufficient for reaction with 1 mg of IgG.
Customizable – vary the molar ratios, reaction buffer and pH, and other parameters to achieve conjugates with different levels of HRP incorporation and activity.
It was by face-to-face visiting when I heard about Abbkine for the first time. It worked while I was doubting about the conjugate efficiency in the beginning! All the graphs fit the results well. In one word, it didn’t let me down. I am considering to purchase from Abbkine again. I really appreciate Abbkine for the wonderful experience it brought to me! I recommend LinKine™ HRP Labeling Kit to those who need it responsibly for it’s high result rate. And I must say that you’ll never regret having it!
2018年7月12日星期四
Abbkine LinKine™ HRP Labeling Kit Review
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.
Read more, please click https://www.nature.com/articles/s41586-018-0137-8
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.
Abbkine LinKine™ HRP Labeling Kit Review
Horseradish peroxidase (HRP) is a 44,173.9-dalton glycoprotein with 6 lysine residues which can be conjugated to a labeled molecule. It produces a colored, fluorometric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified. HRP is often used in conjugates to determine the presence of a molecular target. HRP is also commonly used in techniques such as ELISA and Immunohistochemistry due to its monomeric nature and the ease with which it produces colored products.
Abbkine LinKine™ HRP Labeling Kit is designed for preparing HRP conjugates directly from proteins, peptides, and other ligands that contain a free amino group. The HRP provided in our kit has been pre-activated and can be directly used for conjugation. LinKine™ HRP conjugate Kit contains ready-to-use components to prepare HRP labeled molecules. The resulting conjugates are stable for at least 6 months at 4˚C.
Why should you pick Abbkine LinKine™ HRP Labeling Kit?
Activated HRP – periodate-treated, aldehyde-activated horseradish peroxidase, ready for Labeling to antibodies and other proteins.
High activity HRP – enzyme activity is 120 to 200 units/mg; activated enzyme is stable for at least 12 months at -20°C.
Convenient quantities – each 1 mg-quantity of activated enzyme is sufficient for reaction with 1 mg of IgG.
Customizable – vary the molar ratios, reaction buffer and pH, and other parameters to achieve conjugates with different levels of HRP incorporation and activity.
It was by face-to-face visiting when I heard about Abbkine for the first time. It worked while I was doubting about the conjugate efficiency in the beginning! All the graphs fit the results well. In one word, it didn’t let me down. I am considering to purchase from Abbkine again. I really appreciate Abbkine for the wonderful experience it brought to me! I recommend LinKine™ HRP Labeling Kit to those who need it responsibly for it’s high result rate. And I must say that you’ll never regret having it!
2018年7月6日星期五
Multiplexed precision genome editing with trackable genomic barcodes in yeast
Content introduction:
- High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR–Cas9 in yeast
- Multiplexed precision genome editing with trackable genomic barcodes in yeast
- Secure genome-wide association analysis using multiparty computation
- Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision
- Reversal of siRNA-mediated gene silencing in vivo
1. High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR–Cas9 in yeast
Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, Xiaoge Guo at Wyss Institute for Biologically Inspired Engineering at Harvard University in Boston, Massachusetts, USA and his colleagues introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80–100% in yeast, along with methods for tracking their fitness en masse. They demonstrate the utility of their approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, they created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Their strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.
Read more, please click https://www.nature.com/articles/nbt.4147
2. Multiplexed precision genome editing with trackable genomic barcodes in yeast
Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here Kevin R Roy at Stanford University in Palo Alto, California, USA and his colleagues describe a CRISPR–Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide–donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. They demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA–Fkh1p fusion protein. They performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. They also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.
Read more, please click https://www.nature.com/articles/nbt.4137
3. Secure genome-wide association analysis using multiparty computation
Most sequenced genomes are currently stored in strict access-controlled repositories. Free access to these data could improve the power of genome-wide association studies (GWAS) to identify disease-causing genetic variants and aid the discovery of new drug targets. However, concerns over genetic data privacy may deter individuals from contributing their genomes to scientific studies and could prevent researchers from sharing data with the scientific community. Although cryptographic techniques for secure data analysis exist, none scales to computationally intensive analyses, such as GWAS. Here Hyunghoon Cho at Massachusetts Institute of Technology in Cambridge, Massachusetts, USA and his colleagues describe a protocol for large-scale genome-wide analysis that facilitates quality control and population stratification correction in 9K, 13K, and 23K individuals while maintaining the confidentiality of underlying genotypes and phenotypes. They show the protocol could feasibly scale to a million individuals. This approach may help to make currently restricted data available to the scientific community and could potentially enable secure genome crowdsourcing, allowing individuals to contribute their genomes to a study without compromising their privacy.
Read more, please click https://www.nature.com/articles/nbt.4108
4. Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision
Zehua Bao at University of Illinois at Urbana-Champaign in Urbana, Illinois, USA and his colleagues developed a CRISPR–Cas9- and homology-directed-repair-assisted genome-scale engineering method named CHAnGE that can rapidly output tens of thousands of specific genetic variants in yeast. More than 98% of target sequences were efficiently edited with an average frequency of 82%. They validate the single-nucleotide resolution genome-editing capability of this technology by creating a genome-wide gene disruption collection and apply their method to improve tolerance to growth inhibitors.
Read more, please click https://www.nature.com/articles/nbt.4132
5. Reversal of siRNA-mediated gene silencing in vivo
Ivan Zlatev at Alnylam Pharmaceuticals in Cambridge, Massachusetts, USA and his colleagues report rapid, potent reversal of GalNAc-siRNA-mediated RNA interference (RNAi) activity in vivo with short, synthetic, high-affinity oligonucleotides complementary to the siRNA guide strand. They found that 9-mers with five locked nucleic acids (LNAs) have the highest potency across several targets. Their modular, sequence-specific approach, named REVERSIR, may enhance the therapeutic profile of any long-acting GalNAc–siRNA (short interfering RNA) conjugate by enabling control of RNAi pharmacology.
Read more, please click https://www.nature.com/articles/nbt.4136
2018年7月5日星期四
AbFluor™ 488 Annexin V Apoptosis Detection Kit from Abbkine
Annexin V (or Annexin A5) is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet.
Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells, which can be detected by fluorescence microscopy or flow cytometry.
Propidium iodide (PI) is a fluorescent nucleus dye, impermeant to live cells and early apoptotic cells, but stains late apoptotic, necrotic or dead cells with red fluorescence, binding tightly to the nucleic acids in the cell.
Once binding with DNA, the excitation and emission light of PI-DNA complex is 535 nm and 615 nm.
Abbkine AbFluor™ 488 Annexin V Apoptosis Detection Kit contains Annexin V labeled with Abbkine proprietary green fluorescent dye AbFluor™ 488, which allows the identification and quantitation of apoptotic cells by flow cytometry or fluorescence microscopy. Simultaneous staining of cells with AbFluor™ 488 and propidium iodide (PI) allows the discrimination of intact cells, early apoptotic and late apoptotic or necrotic cells.
[caption id="attachment_84161" align="aligncenter" width="635"]
Abbkine AbFluor™ 488 dye is better than FITC, Alexa Fluor 488, Dylight 488 as it is brighter, not affected by pH, and has much better photostability.
Below is Abbkine Annexin V Apoptosis Detection Kits details:
[table id=15 /]
FAQ
1. Can Abbkine Annexin V Apoptosis Detection Kit detect apoptosis in animals other than humans?
Sure. Because annexin v is compatible with phosphatidylserine ( PS ), but PS has no difference among different species.
2. What’s the effect of pancreatin on adherent cells?
Pancreatin has some damage to the cell membrane. But when digested with low concentration pancreatin, the adherent cells are gently blown 2 to 3 times, centrifuged at 4 , 300 g, and 5 min, the damage caused by pancreatin can be controlled within 5 %. And there is no obvious influence on the experimental results when there is a control group.
3. Why can only use pancreatin without EDTA to digest adherent cells?
Annexin V is a Ca+ dependent protein, so EDTA cannot be added to prevent EDTA from sequestering Ca+, thus affecting annexin V and the results.
