How Does Western Blotting Work

Western blotting is a technique in molecular biology used in the detection of a specific protein within a sample. It uses SDS-PAGE to separate proteins based on their size and these separated proteins are then transferred into a membrane. This technique uses specific primary antibodies in the labeling of a particular protein in the blot. Secondary antibodies that are labeled with reporter proteins detect the labled proteins with the primary antibody.

Key Areas Covered

1. What is Western Blotting
     – Definition, Facts, Applications
2. How Does Western Blotting Work
     – Process of Western Blotting

Key Terms: Color Development, Nitrocellulose Membrane, Primary Antibody, Secondary Antibody, Western Blotting 

How Does Western Blotting Work_Infographic

What is Western Blotting

Western blotting is a hybridization technique used in the detection of a particular protein within a sample. It is also called immunoblotting since the technique uses antibodies for both labeling the protein of interest and the detection of the antibody-labeled proteins.

How Does Western Blotting Work_Figure 1

Figure 1: Western Blot

Western blotting was first developed by Towbin in 1979, and it is currently used as a routine technique of protein analysis. 

How Does Western Blotting Work

Western blotting is mainly used in the detection of a specific protein in a sample. The steps of the Western blotting technique are described below.

  1. SDS-PAGE – The protein sample is separated based on their size by SDS-PAGE.
  2. Transferring proteins to a membrane – The size-fractionated proteins are transferred into either a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. The techniques involved in this transfer is diffusion transfer.
  3. Blocking non-specific sites – The unoccupied sites on the membrane are blocked to prevent the non-specific binding of proteins. Nonfat dry milk or Bovine serum albumin (BSA) can be used for this purpose.
  4. Primary antibody incubation – The blocked membrane is incubated with a primary antibody solution. These primary antibodies bind to a particular protein on the membrane.
  5. Secondary antibody incubation – The membrane is incubated with secondary antibodies, which binds to the primary antibodies. Secondary antibodies are conjugated to reporter proteins. These reporter proteins can be biotin, alkaline phosphatase or horseradish peroxidase.
  6. Protein detection by color development – The conjugated enzyme reporters react with their substrate to generate a colored Alkaline phosphatase converts BCIP to a blue color product while horseradish peroxidase converts luminol, emitting luminesce.
How Does Western Blotting Work

Figure 2: Western Blotting – Procedure


Western blotting is a hybridization technique used in the detection of the presence of a particular protein within a sample. It uses SDS-PAGE to separate proteins based on their size and binding with primary antibodies, which can be recognized by secondary antibodies during color development.


1. “Western Blotting Fundamental Principle, How Western Blots Work.” Boster, .

Image Courtesy:

1. “Anti-lipoic acid immunoblot” By TimVickers – Own work via
2. “Western Blotting” By Cawang – Own work, CC0) via

About the Author: Lakna

Lakna, a graduate in Molecular Biology & Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things

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