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[Home](https://www.sigmaaldrich.com/US/en)[Applications](https://www.sigmaaldrich.com/US/en/applications)[Protein Biology](https://www.sigmaaldrich.com/US/en/applications/protein-biology)Western Blotting
# Western Blotting
Western blotting is a well-established analytical technique for detecting, analyzing, and quantifying proteins. This method is widely used to detect specific protein molecules in complex samples such as tissue homogenates and cell lysates. Western blotting typically involves protein separation by gel electrophoresis followed by transfer to a polyvinylidene difluoride (PVDF) or nitrocellulose membrane. After proteins have been transferred, they can be stained for visualization and directly identified by N-terminal sequencing, mass spectrometry or immunodetection.
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Overview
Related Articles & Protocols
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In Western blotting immunodetection, proteins are identified through their binding to specific antibodies. Typically, a primary antibody is used in combination with an HRP- or AP-conjugated secondary antibody for chemiluminescent or colorimetric detection using an appropriate substrate. Alternatively, a fluorescently labeled primary or secondary antibody can be used for direct visualization.
Western blotting is used extensively in biochemistry to detect the presence of specific proteins, to determine the extent of post-translational modifications, to verify protein expression in cloning applications, to analyze protein and biomarker expression levels, in antibody epitope mapping, and to test for markers of disease in clinical settings.
The need to simultaneously analyze more proteins in limited samples has driven ongoing research into improving the sensitivity and speed of blotting techniques. Double blotting eliminates false positives caused by nonspecific interactions. Far-Western blotting enables the detection of specific protein-protein interactions. Southwestern blotting is used to identify proteins that interact with specific DNA sequences. Multistrip blotting increases throughput while minimizing inter-blot variability. New technologies are being developed to reduce the amounts of protein required to produce a signal and improve the quantitative capabilities of Western blotting.
## Workflow
### Protein Extraction
When working with cells or tissues, samples must be disrupted to isolate the proteins prior to analysis. Lysis and extraction buffers use detergents to break down cell walls and membranes to release the proteins. The optimal buffer should be chosen based on the sample type and sub-cellular localization of the target protein.
- [Sample Preparation for Western blotting](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/western-blotting/sample-preparation)
### Gel Electrophoresis
Protein gel electrophoresis is used to separate and resolve proteins prior to blotting. The prepared protein mixture is run on a polyacrylamide gel to sort proteins by molecular weight and charge.
- [Introduction to SDS-PAGE](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/gel-electrophoresis/sds-page)
- [Running an mPAGE® Gel with an XCell SureLock® System](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/gel-electrophoresis/running-protein-gel-invitrogen)
- [Running an mPAGE® Gel with a Bio-Rad Tank](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/gel-electrophoresis/running-protein-gel-biorad)
- [Troubleshooting Gel Electrophoresis](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/gel-electrophoresis/troubleshooting-tables)
### Transfer to a Membrane
The proteins separated on the gel by electrophoresis are immobilized by transfer onto a PVDF or nitrocellulose membrane. The transfer uses electric current to pull proteins from the gel onto the membrane (electroblotting).
- [Blotting Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/western-blotting/western-blotting)
- [Semi-dry or Tank Transfer](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/semi-dry-tank-electrotransfer)
### Detection
Target protein is detected using a primary antibody in combination with an HRP- or AP-conjugated secondary antibody and an appropriate chemiluminescent or colorimetric substrate, or by using fluorescently-labeled primary or secondary antibody.
- [Antibody Basics](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/antibody-basics)
- [How to Choose a Secondary Antibody](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/secondary-antibody-selection)
- [Immunodetection Protocols](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/western-blotting/western-blotting#traditional)
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## Related Articles
- [Antibody Basics](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/antibody-basics)
Explore the basics of working with antibodies including technical information on structure, classes, and normal immunoglobulin ranges.
- [Alkaline Phosphatase](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/alkaline-phosphatase)
We offer a broad range of alkaline phosphatase enzymes and substrates that are optimized for conjugation to antibodies and other proteins for your specific application needs.
- [Loading Controls for Western Blotting](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/loading-controls-western-blotting)
Loading controls in western blotting application.
- [Gelatine](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/gelatin)
Gelatin is a heterogeneous mixture of water-soluble proteins extracted by boiling skin, tendons, ligaments, bones, etc. in the water.
- [Semi-Dry or Tank Electrotransfer Troubleshooting](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/semi-dry-tank-electrotransfer)
Troubleshoot protein transfer issues in Western blotting by understanding causes and optimizing transfer conditions.
- [See All (33)](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search)
## Related Protocols
- [Cell Lysis and Protein Extraction for Western Blotting](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/western-blotting/sample-preparation)
Protocol for sample preparation for cell lysis and efficient protein extraction from cultured tissues and cells for subsequent Western blotting.
- [Western Blot Protocol - Immunoblotting or Western Blot](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/western-blotting/western-blotting)
Western blot protocol details protein transfer from gels to nitrocellulose, crucial for immunoblotting procedures in research.
- [Immunodetection Using BCIP/NBT Substrate](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/western-blotting/bcip-nbt-immunodetection)
BCIP/NBT substrate produces stable, intense color for alkaline phosphatase assays, ideal for visual observation.
- [Chemiluminescence Detection with Amersham ECL, Amersham ECL Prime, and Amersham ECL Select](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/western-blotting/chemiluminescence-detection)
This page describes chemiluminescence detection of GST-tagged proteins with Amersham ECL, Amersham ECL Prime, and Amersham ECL Select from Cytiva.
- [30-minute Immunodetection Protocol Using the SNAP i.d.® 2.0 System](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/western-blotting/30-minute-immunodetection-protocol)
The SNAP i.d. 2.0 Protein Detection System is the second generation of the SNAP i.d. method for detecting immunoreactive proteins on Western blots.
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__Featured Articles__
[Antibody Basics](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/antibody-basics)
Explore the basics of working with antibodies including technical information on structure, classes, and normal immunoglobulin ranges.
[Alkaline Phosphatase](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/alkaline-phosphatase)
We offer a broad range of alkaline phosphatase enzymes and substrates that are optimized for conjugation to antibodies and other proteins for your specific application needs.
[Loading Controls for Western Blotting](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/loading-controls-western-blotting)
Loading controls in western blotting application.
[Gelatine](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/western-blotting/gelatin)
Gelatin is a heterogeneous mixture of water-soluble proteins extracted by boiling skin, tendons, ligaments, bones, etc. in the water.
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