Reversal of indoleamine 2,3-dioxygenase–mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme

Content introduction:

• Reversal of indoleamine 2,3-dioxygenase–mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme


• Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements


• Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip


• Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling


• A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

1. Reversal of indoleamine 2,3-dioxygenase–mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here Todd A Triplett at University of Texas at Austin (UT Austin) in Austin, Texas, USA and his colleagues show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. They show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.



Read more, please click https://www.nature.com/articles/nbt.4180

2. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

CRISPR–Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR–Cas9 was reasonably specific. Here Michael Kosicki at Wellcome Sanger Institute in Hinxton, UK and his colleagues report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, they show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.

Read more, please click https://www.nature.com/articles/nbt.4192

3. Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip

The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. Arjang Hassibi at InSilixa, Inc. in Sunnyvale, California, USA and his colleagues developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. They detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. They also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.

Read more, please click https://www.nature.com/articles/nbt.4179

4. Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here Maria D Giraldez at University of Michigan in Ann Arbor, Michigan, USA and her colleagues report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. They assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. They found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

Read more, please click https://www.nature.com/articles/nbt.4183

5. A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

The neurotransmitter acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body. Despite its importance, cholinergic transmission in the majority of tissues and organs remains poorly understood owing primarily to the limitations of available ACh-monitoring techniques. Miao Jing at Peking University School of Life Sciences in Beijing, China and her colleagues developed a family of ACh sensors (GACh) based on G-protein-coupled receptors that has the sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from multiple animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescence, confocal, and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing-pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable tool for monitoring cholinergic transmission underlying diverse biological processes.

Read more, please click https://www.nature.com/articles/nbt.4184

Reversal of indoleamine 2,3-dioxygenase–mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme

Content introduction:

• Reversal of indoleamine 2,3-dioxygenase–mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme


• Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements


• Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip


• Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling


• A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

1. Reversal of indoleamine 2,3-dioxygenase–mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here Todd A Triplett at University of Texas at Austin (UT Austin) in Austin, Texas, USA and his colleagues show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. They show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.



Read more, please click https://www.nature.com/articles/nbt.4180

2. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

CRISPR–Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR–Cas9 was reasonably specific. Here Michael Kosicki at Wellcome Sanger Institute in Hinxton, UK and his colleagues report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, they show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.

Read more, please click https://www.nature.com/articles/nbt.4192

3. Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip

The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. Arjang Hassibi at InSilixa, Inc. in Sunnyvale, California, USA and his colleagues developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. They detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. They also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.

Read more, please click https://www.nature.com/articles/nbt.4179

4. Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here Maria D Giraldez at University of Michigan in Ann Arbor, Michigan, USA and her colleagues report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. They assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. They found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

Read more, please click https://www.nature.com/articles/nbt.4183

5. A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

The neurotransmitter acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body. Despite its importance, cholinergic transmission in the majority of tissues and organs remains poorly understood owing primarily to the limitations of available ACh-monitoring techniques. Miao Jing at Peking University School of Life Sciences in Beijing, China and her colleagues developed a family of ACh sensors (GACh) based on G-protein-coupled receptors that has the sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from multiple animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescence, confocal, and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing-pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable tool for monitoring cholinergic transmission underlying diverse biological processes.

Read more, please click https://www.nature.com/articles/nbt.4184

Popular anti-HA epitope tag antibodies || Abbkine

The last time we presented anti-DDDDK tag antibodies, today I will overview anti-HA epitope tag antibodies for a variety of research needs.

We know that Human influenza hemagglutinin (HA) is a surface glycoprotein required for the infectivity of the human virus. The HA tag is derived from the HA-molecule corresponding to amino acids 98-106 and has been extensively used as a general epitope tag in expression vectors.

Abbkine not only offers highly specific monoclonal and polyclonal anti-HA tag antibodies, but also provides AbFluor 350, 405, 488, 555, 594, 647, 680, Cy3, Cy5, FITC, agarose, magnetic beads and HRP conjugated anti-HA tag antibodies.

You can always find a product that best suits your experiment below.
[table id=17 /]

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.

Anti-His tag antibodies will be continued next time.

Popular anti-HA epitope tag antibodies || Abbkine

The last time we presented anti-DDDDK tag antibodies, today I will overview anti-HA epitope tag antibodies for a variety of research needs.

We know that Human influenza hemagglutinin (HA) is a surface glycoprotein required for the infectivity of the human virus. The HA tag is derived from the HA-molecule corresponding to amino acids 98-106 and has been extensively used as a general epitope tag in expression vectors.

Abbkine not only offers highly specific monoclonal and polyclonal anti-HA tag antibodies, but also provides AbFluor 350, 405, 488, 555, 594, 647, 680, Cy3, Cy5, FITC, agarose, magnetic beads and HRP conjugated anti-HA tag antibodies.

You can always find a product that best suits your experiment below.
[table id=17 /]

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.

Anti-His tag antibodies will be continued next time.

Popular anti-HA epitope tag antibodies || Abbkine

The last time we presented anti-DDDDK tag antibodies, today I will overview anti-HA epitope tag antibodies for a variety of research needs.

We know that Human influenza hemagglutinin (HA) is a surface glycoprotein required for the infectivity of the human virus. The HA tag is derived from the HA-molecule corresponding to amino acids 98-106 and has been extensively used as a general epitope tag in expression vectors.

Abbkine not only offers highly specific monoclonal and polyclonal anti-HA tag antibodies, but also provides AbFluor 350, 405, 488, 555, 594, 647, 680, Cy3, Cy5, FITC, agarose, magnetic beads and HRP conjugated anti-HA tag antibodies.

You can always find a product that best suits your experiment below.
[table id=17 /]

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.

Anti-His tag antibodies will be continued next time.

Western blot protocol

Western blot (sometimes called the protein immunoblot) is a widely used analytical technique used in molecular biology, immunogenetics and other molecular biology disciplines to detect specific proteins in a sample of tissue homogenate or extract. The typical western blot protocol that Abbkine antibodies are subjected to is reproduced below.
Note: Some of the steps in this protocol require optimization depending on the sample and antibody being used.

1. Sample preparation

Protein can be extracted from different kind of samples, such as tissue or cells. Lysis buffers differ in their ability to solubilize proteins, with those containing sodium dodecyl sulfate (SDS) and other ionic detergents considered to be the harshest and therefore most likely to give the highest yield.
As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down significantly if samples are kept on ice or at 4°C at all times and appropriate inhibitors are added fresh to the lysis buffer. To denature, use a loading buffer with the anionic detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95–100°C for 5 min. Heating at 70°C for 5–10 min is also acceptable and may be preferable when studying multi-pass membrane proteins. These tend to aggregate when boiled and the aggregates may not enter the gel efficiently.

2. SDS-PAGE

The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to identify a protein.
By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and SH]) and thus allows separation of proteins by their molecular mass. Sampled proteins become covered in the negatively charged SDS, effectively becoming anionic, and migrate towards the positively charged (higher voltage) anode (usually having a red wire) through the acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh, and the proteins are thus separated according to size (usually measured in kilodaltons, kDa). The concentration of acrylamide determines the resolution of the gel - the greater the acrylamide concentration, the better the resolution of lower molecular weight proteins. Typically, 10–50 µg of protein is loaded per well depending on protein abundance, molecular weight, and type of gel used.

Figure 1 The gel percentage required for the size of proteins

3. Transfer

To make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The primary method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. An older method of transfer involves placing a membrane on top of the gel, and a stack of filter papers on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. In practice this method is not used as it takes too much time; electroblotting is preferred, in which case, as with PAGE-SDS, proteins migrate toward the (+) anode (red wire on most instruments). As a result of either "blotting" process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings.

Figure 2 Western blot transfer

4. Blocking

Since the membrane has been chosen for its ability to bind protein and as both antibodies and the target are proteins, steps must be taken to prevent the interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein – typically 3–5% bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in tris-buffered saline (TBS) or I-Block, with a minute percentage (0.1%) of detergent such as Tween 20 or Triton X-100. Although non-fat dry milk is preferred due to its availability, an appropriate blocking solution is needed as not all proteins in milk are compatible with all the detection bands. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces background in the final product of the western blot, leading to clearer results, and eliminates false positives.

5. Probing with primary and secondary antibodies

During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme; when exposed to an appropriate substrate, this enzyme drives a colorimetric reaction and produces a color. For a variety of reasons, this traditionally takes place in a two-step process, although there are now one-step detection methods available for certain applications. The amount of primary and secondary antibody will vary depending on the antibody being used and the samples you are running. A good starting point is to check the antibody product pages for recommended dilution ranges and from there titrate to find the optimal antibody dilution for the experiment.

6. Developing the blot

After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns reveal protein only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is commonly repeated for a structural protein, such as actin or tubulin, that should not change between samples. The amount of target protein is normalized to the structural protein to control between groups. A superior strategy is the normalization to the total protein visualized with trichloroethanol or epicocconone. This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers.

HRP western blot
Develop the blot using ECL Chemiluminescent Substrate (K22020 SuperLumia ECL HRP Substrate Kit) for 1min. Place the membrane in clear film and remove any excess substrate. If the exposure time is longer than a few min, consider using a more sensitive substrate, such as (K22030 SuperLumia ECL Plus HRP Substrate Kit).
Fluorescent western blot
Acquire image using a fluorescent imager. Place the blot on the imaging surface and ensure it is lying flat with no air bubbles. If you are multiplexing, use the 800 nm channel for detection of less abundant proteins or weak targets. Use the 680 nm channel for detection of more abundant proteins or strong targets.

Western blot protocol

Western blot (sometimes called the protein immunoblot) is a widely used analytical technique used in molecular biology, immunogenetics and other molecular biology disciplines to detect specific proteins in a sample of tissue homogenate or extract. The typical western blot protocol that Abbkine antibodies are subjected to is reproduced below.
Note: Some of the steps in this protocol require optimization depending on the sample and antibody being used.

1. Sample preparation

Protein can be extracted from different kind of samples, such as tissue or cells. Lysis buffers differ in their ability to solubilize proteins, with those containing sodium dodecyl sulfate (SDS) and other ionic detergents considered to be the harshest and therefore most likely to give the highest yield.
As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down significantly if samples are kept on ice or at 4°C at all times and appropriate inhibitors are added fresh to the lysis buffer. To denature, use a loading buffer with the anionic detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95–100°C for 5 min. Heating at 70°C for 5–10 min is also acceptable and may be preferable when studying multi-pass membrane proteins. These tend to aggregate when boiled and the aggregates may not enter the gel efficiently.

2. SDS-PAGE

The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to identify a protein.
By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and SH]) and thus allows separation of proteins by their molecular mass. Sampled proteins become covered in the negatively charged SDS, effectively becoming anionic, and migrate towards the positively charged (higher voltage) anode (usually having a red wire) through the acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh, and the proteins are thus separated according to size (usually measured in kilodaltons, kDa). The concentration of acrylamide determines the resolution of the gel - the greater the acrylamide concentration, the better the resolution of lower molecular weight proteins. Typically, 10–50 µg of protein is loaded per well depending on protein abundance, molecular weight, and type of gel used.

Figure 1 The gel percentage required for the size of proteins

3. Transfer

To make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The primary method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. An older method of transfer involves placing a membrane on top of the gel, and a stack of filter papers on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. In practice this method is not used as it takes too much time; electroblotting is preferred, in which case, as with PAGE-SDS, proteins migrate toward the (+) anode (red wire on most instruments). As a result of either "blotting" process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings.

Figure 2 Western blot transfer

4. Blocking

Since the membrane has been chosen for its ability to bind protein and as both antibodies and the target are proteins, steps must be taken to prevent the interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein – typically 3–5% bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in tris-buffered saline (TBS) or I-Block, with a minute percentage (0.1%) of detergent such as Tween 20 or Triton X-100. Although non-fat dry milk is preferred due to its availability, an appropriate blocking solution is needed as not all proteins in milk are compatible with all the detection bands. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces background in the final product of the western blot, leading to clearer results, and eliminates false positives.

5. Probing with primary and secondary antibodies

During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme; when exposed to an appropriate substrate, this enzyme drives a colorimetric reaction and produces a color. For a variety of reasons, this traditionally takes place in a two-step process, although there are now one-step detection methods available for certain applications. The amount of primary and secondary antibody will vary depending on the antibody being used and the samples you are running. A good starting point is to check the antibody product pages for recommended dilution ranges and from there titrate to find the optimal antibody dilution for the experiment.

6. Developing the blot

After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns reveal protein only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is commonly repeated for a structural protein, such as actin or tubulin, that should not change between samples. The amount of target protein is normalized to the structural protein to control between groups. A superior strategy is the normalization to the total protein visualized with trichloroethanol or epicocconone. This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers.

HRP western blot
Develop the blot using ECL Chemiluminescent Substrate (K22020 SuperLumia ECL HRP Substrate Kit) for 1min. Place the membrane in clear film and remove any excess substrate. If the exposure time is longer than a few min, consider using a more sensitive substrate, such as (K22030 SuperLumia ECL Plus HRP Substrate Kit).
Fluorescent western blot
Acquire image using a fluorescent imager. Place the blot on the imaging surface and ensure it is lying flat with no air bubbles. If you are multiplexing, use the 800 nm channel for detection of less abundant proteins or weak targets. Use the 680 nm channel for detection of more abundant proteins or strong targets.