Western Blot
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- Western Blot Introduction
What's western blot test? Western blot test is a technique used to detect specific proteins from samples such as cell extracts or tissue homogenates, also analyze recombinant proteins synthesized in vitro. Western blot can also be used to identify a target protein on the basis of the special affinity between a certain antigen and its relative antibody. Western blot generally contains three main steps, SDS-PAGE firstly, then blot of samples, and finally immunology test. Qualitative and semi-quantitative data can be acquired about the target protein through western blot. If both qualitative and quantitative data is wanted, another technique ELISA is suggested. Learn more about the traits comparison between western blot and other methods like ELISA, IHC/ICC, and IFA (Immunofluorescence assay). Besides, about western blot test, it's necessary to mention that the name "Western blot " is a play on the name of southern blot test, which is named after its inventor Edwin Southern. In the field of protein, western blot is widely used for the test of protein expression level.
Detailed workflow of western blot varied widely, as there are two methods for western blot detection, indirect vs. direct detection. In the indirect detection of western blot, a primary antibody is added first to bind to the epitope of the target protein; the primary antibody is then recognized by relative secondary antibody labeled with enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). Other labels, for example, biotin, fluorescent probes such as fluorescein or rhodamine are also included. Learn more about western blot workflow (for indirect detection of western blot). While with the direct detection of western blot, just a primary antibody labeled with enzyme or fluorescent dye is needed, without any use of secondary antibody. However, most researchers prefer indirect detection of western blot in most cases due to a variety of advantages over direct detection of western blot. Learn more about the comparison between indirect and direct detection methods.
This website of western blot is divided into several sections starting with a western blot introduction. This is followed by more specific sections discussing the factors which need to be addressed in order to develop a successful western blot, detailed western blot protocols described according to western blot workflow, and tips that will help improve the precision of an assay. The section of western blot troubleshooting provides information to help solve almost all the problems encountered in a western blot. References give more information or links to a further understanding of western blot.
- Western Blot Protocol
Protein Expression
Expression System,
Protein Stability Assay,
Protein Production
Western Blot Buffer
Loading Buffer,
Running Buffer, Transfer Buffer,
Blocking Buffer, Washing Buffer
Sample Preparation
RIPA Lysis Buffer,
NP40 Lysis Buffer,
BCA Method
SDS-PAGE
Gel Preparation,
Resolving Gel Solution,
Stacking Gel Solution
Protein Transfer
Semi-dry Transfer,
Wet Transfer,
PVDF, Nitrocellulose,
Transfer Efficiency
Membrane Blocking
Blocking Solution,
Blocking Principle,
Blocking Method
Antibody Incubation
Primary Antibody, MAb,
PAb, Secondary Antibody,
Incubation Buffer, Time, Temp.
Antibody detection
Indirect vs. Direct Detection,
Chemiluminescence, Fluorescence,
Chemical Coloration
- Western Blot Troubleshooting
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·No Bands (Signal) |
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- Antibody for Western Blot
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Rabbit MAb, Mouse MAb, etc. |
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Rabbit PAb, etc. |
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- References for Western Blot
E.M. Southern, Detection of specific sequences among DNA fragments separated by gel electrophoresis, Journal of Molecular Biology, Volume 98, Issue 3, 5 November 1975, Pages 503-517, ISSN 0022-2836, DOI: 10.1016/S0022-2836(75)80083-0.
W. Neal Burnette, 'Western Blotting': Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A, Analytical Biochemistry, Volume 112, Issue 2, April 1981, Pages 195-203, ISSN 0003-2697, DOI: 10.1016/0003-2697(81)90281-5.
"Imaging Systems for Westerns: Chemiluminescence vs. Infrared Detection, 2009 - , Methods in Molecular Biology, Protein Blotting and Detection, vol. 536". Humana Press. Retrieved 2010-20-26.
Burnette WN. (1981). "'Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A". Analytical Biochemistry 112 (2): 195–203. DOI: 10.1016/0003-2697(81)90281-5.
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