Western Blotting (WB) Methods, Techniques & Protocols




Definition: technique which transfers proteins from a gel to a nitrocellulose or nylon membrane following electrophoresis and uses specific antibodies to bind and visualize the protein of interest. It is a technique for analyzing and identifying protein antigens: the proteins are separated by electrophoresis in polyacrylamide gel, then transferred ("blotted") onto a nitrocellulose membrane or treated paper, where they bind in the same pattern as they formed in the gel. The antigen is overlaid first with antibody, then with anti-immunoglobulin or protein A labeled with radioisotope, fluorescent dye, or enzyme (source: NDI Foundation)

General Methods & Techniques

Antigen-Antibody Specific Applications

Virological Applications

Tumor, Disease & Diagnostic Applications

Western Blot From Wikipedia, the free encyclopedia - A western blot (a.k.a immunoblot) is a method in molecular biology/biochemistry/immunogenetics to detect protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate denatured proteins by mass. The proteins are then transferred out of the gel and onto a membrane (typically nitrocellulose), where they are "probed" using antibodies specific to the protein. As a result, researchers can examine the amount of protein in a given sample and compare levels between several groups.

Introduction to Western Blot Activity - This exercise was developed in conjuction with Dr. Douglas Lake of the University of Arizona School of Medicine's Department of Microbiology and Immunology. As part of the second year medical student Immunology curricula, this demanding material is designed to expose future doctors to laboratory procedures essential to modern clinical practice.

Western Blotting Technique by Mama Ji - Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the size of the protein. It does not matter whether the protein has been synthesized in vivo or in vitro. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. So you must be able to produce at least a small portion of the protein from a cloned DNA fragment. You will use this antibody as a probe to detect the protein of interest.

Introduction to Western Blotting (PIERCE) - The term “blotting” refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. Western blotting (also called immunoblotting because an antibody is used to specifically detect its antigen) was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis. The specificity of the antibody-antigen interaction enables a single protein to be identified in the midst of a complex protein mixture. Western blotting is commonly used to positively identify a specific protein in a complex mixture and to obtain qualitative and semiquantitative data about that protein.

Enzyme-Assisted Immunoelectroblotting (IEB OR Western Blotting) - Molecular Biology Techniques Manual, Third Edition, Edited by: Vernon E Coyne, M Diane James, Sharon J Reid and Edward P Rybicki.

Western Blot Procedure - This is a brief overview of how a western blot (more formally called a protein immunoblot) is performed and what type of data you can obtain from one.

Western Protocol from Stephen B. Howell's Lab, University of California, San Diego - Detailed Western Blotting protocol inlcuding preparation of cell lysates, polyacrylamide gel, preparation of gel, running the gel, preparation of membrane, antibodies and detection, buffers, etc.

Western Blotting Technique and Protocol - Western blot is a powerful technique in that; it leads to simultaneously detection of a specific protein by means of its antigenicity and its molecular mass. The proteins are first separated by mass using SDS-PAGE, transferred from gels onto membranes and then specifically detected in the immunoassay step.

Far Western Protocol from Krause Lab, University of Toronto - This protocol is used in two recent publications (Guichet et al,1997; Schwartz et al, 2001).

Western Blotting with Monoclonal Antibodies, BD Biosciences - A detailed step by step protocol.

Is a Positive Western Blot Proof of HIV Infection? - It is currently accepted that a positive Western blot (WB) HIV antibody test is synonymous with HIV infection and the attendant risk of developing and dying from AIDS. In this communication we present a critical evaluation of the presently available data on HIV isolation and antibody testing.

Immunoblotting Protocol (eBioscience) - The immunoblotting technique provides information about presence, molecular weight, and/or quantity of an antigen by combining protein separation via gel electrophoresis with specific recognition of antigens by antibodies. Immunoblotting is useful when the antigen of interest is insoluble or readily degraded and cannot be easily immunoprecipitated. Since most gel electrophoresis procedures result in denaturation of the antigen, only polyclonal and monoclonal antibodies that recognize the denatured form of an antigen can be utilized in immunoblotting. To study proteins that are expressed at very low levels, it is recommended that immunoprecipitation be followed by immunoblotting for more sensitive detection.

Protocol for Western Blotting of Tissue Culture Cells

Western Blotting with Biotinylated Antibodies (BD Biosciences) - A detailed step by step protocol.

Introduction to Antibodies - Protein (Western) Blotting (Chemicon) - Immunoblotting procedures combine the resolution of gel electrophoresis with the specificity of antibody detection. Blotting can be used to ascertain a number of important characteristics of protein antigens, including the presence and quantity of an antigen, the molecular weight of the antigen, and the efficiency of antigen extraction. This method is especially helpful when dealing with antigens that are insoluble, difficult to label, or easily degraded, and thus not amenable to procedures such as immunoprecipitation.

Kamps's Western Blotting Protocol - ECL is an appealing technique because it is quick and very sensitive and does not expose the investigator to radioactivity. The use of radioiodinated protein A to detect bound antibodies is however usually a superior technique.

Western Blot and Gel Overlays - I illustrate Western blots and gel overlayson this page. See Old and Primrose for details.

Western Blot Protocol (Frank Lab, Oklahoma Medical Research Foundation) - In a Western blot, proteins that are separated on polyacrylamide gels on the basis of size are transferred to a membrane for detection with antibodies.

Western Blot Overview, Protocol, and Troubleshooting (ProSci) - A Western Blot (also known as an ImmunoBlot) is a means of detecting the presence of a specific protein within a tissue or mixture. It can also be used to determine the quantity and molecular weight of the protein of interest.

How to do a Western Blot (Jonathan Flint, UK) - A detailed western blotting method including reagents, lysis, Quantitation, Making a Gel. Gel Running, Transfer, Blotting, Stripping.

Western Blotting Using the SemiPhor Semi-Dry Transfer Unit - 1. Remove the stacking gel from the gel. Measure the gel and record its size. Do not pre-soak the gel. Do not cut a corner off the gel, as this may allow a short circuit and inefficient transfer. Blot the gel as soon as possible after electrophoresis to avoid diffusion of the samples through the gel.

What is protein blotting? A website dedicated to Western Blotting Technique - Protein blotting is an analytical method that involves the immobilization of proteins on membranes before detection using monoclonal or polyclonal antibodies. There are different blotting protocols (dot blot, 2D blot), however one of the most powerful is western blotting.