Western Blotting

By Parth Kumar | 5 September 2024

Western Blotting is an essential step in protein purification. It involves the transfer of proteins from a gel medium to a membrane. SDS-PAGE precedes Western Blotting, and is used to separate proteins in a sample based on their molecular weight, under the influence of an electric field parallel to the gel surface. In contrast, Western Blotting is the transfer of separated/resolved proteins (by SDS-PAGE) to a membrane, by an electric field perpendicular to gel surface - causing proteins to move out into the membrane.

Why is Western Blotting important? It helps in the detection specific proteins in a blood or tissue sample. THis is known as probing. It is a fundamental step in the separation and identification of proteins in confirmatory medical diagnoses (such as HIV and Lyme Disease), as well as for protein localisation in cells. it is a more specific technique than ELISA (Enzyme-Linked Immunosorbent Assay).

Interestingly, the name Western Blotting comes from a similar technique used for the transfer of DNA strands - called Southern Blotting after the scientist Robert Southern. The analogous techniques for RNA and proteins were simply named Northern and Western - and not because they were after scientists by those names!

The membrane (on to which proteins are transferred) is between the gel surface and positive electrode in a sandwich. After SDS-PAGE, all the proteins have already acquired a uniform negative charge. A fiber pad or sponge is kept at positive end (the top) and filter papers are used to protect the gel and blotting membrane. Two things are important here - close contact of the gel and the membrane (to get a clear image), and the correct placement and orientation of the membrane between the gel and the positive electrode.

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Western Blotting is followed up with immunostaining to identify where the protein of interest is. In simple words, it makes use of the specificity and fidelity of antigen-antibody interactions. An antibody specific to the protein of interest is made to bind with it, and a secondary antibody binds to the primary antibody and either gives of a fluorescent or radioactive signal (autoradiography) or is conjugated with an enzyme which can be quantified using an assay. For instance, the enzyme horseradish peroxidase acts on substrate TMB in the presence of hydrogen peroxide to give a bright blue oxidised product.

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The membrane used in Western Blotting is broadly of two types:

  1. PVDF (Polyvinylidene Difluoride): has better mechanical support, allows reprobing and storage, but background noise is higher which necessitates good washing
  2. Nitrocellulose (used in Southern Blotting too): has high affinity to protein, high retention ability, but brittle, and doesn’t allow reprobing

Reprobing is the process of removing primary and secondary antibodies used for immunostaining and carrying out immunostaining again with different antibodies to detect additional proteins of interest.

Blocking is an essential step in Western Blotting and immunostaining. It blocks the sites on the membrane not occupied by proteins, to prevent non-specific antibody binding. This is usually done with 5% BSA (bovine serum albumin, a protein) or casein (a milk protein)

References:

  1. Mahmood, T., & Yang, P. C. (2012). Western blot: technique, theory, and trouble shooting. North American journal of medical sciences, 4(9), 429–434.
  2. Image: MyBioSource

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