Western blotting is a multi-step procedure that utilizes highly selective antibodies that bind to a specific protein of interest, allowing investigators to detect a specific protein in a complex mixture of proteins.
When you use suitable antibodies, buffers and detection methods, you will reveal the secrets of your protein samples.
Download this guide to discover tips on:
- Optimizing the gel and loading samples
- Antibody validation and transfer conditions
- Buffer preparation and staining
How To Guide
Mastering the Western Blotting
Frontier: Advanced Tips and Tricks
for Success
Aldrin V. Gomes
If you had to pick a few techniques to use in a laboratory, western blotting would most likely be one of
those techniques. If you love puzzles, you probably love western blotting. The technique is like solving
a mystery with clues hidden in the gel, antibodies, buffers and the membrane. When you use suitable
antibodies, buffers and detection methods, you will reveal the secrets of your protein samples. On June
28, 2024, a PubMed search for “western blot” returned 413,000 results, indicating its immense popularity.
This blotting technique’s affordability and adaptability have contributed significantly to its use in biological
research compared to other methods. Western blotting, or immunoblotting, is simply a multi-step pro
cedure
that utilizes highly selective antibodies that bind to a specific protein of interest, allowing inves
tigators
to detect a specific protein in a complex mixture of proteins.1 Besides determining if a protein of
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interest is present in a sample, western blotting can also allow you to determine the amount of protein,
the molecular weight of the protein and detect modifications on proteins.1 The most common protein
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modifications that can be detected include phosphorylation, glycosylation, ubiquitination, acetylation and
methylation. The key to detecting protein modifications is having an antibody specific for the modificatio
on the protein of interest.
The main steps of a western blotting experiment include sample preparation, applying the sample to a
polyacrylamide gel, which separates proteins based on size, protein transfer from the polyacrylamide gel
to a membrane (typically nitrocellulose or polyvinylidene fluoride (PVDF)), blocking non-specific binding
sites on the membrane, incubation of the membrane with an antibody that is specific for the protein of
interest, incubation of the membrane with secondary antibodies that interact with the primary antibody
and detection of the signal generated by the secondary antibody.
While western blotting is one of my favorite techniques, it has two main downsides: the high dependency
of the method on the availability of a specific antibody and the thousands of possible variations of pro
tocols
for the technique. To help reduce the variability in procedures for new users, we have a detailed
protocol that has been optimized over 15 years — it is available on protols.io.2 While a quick search online
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can find tips for each step of the western blotting procedure, I will discuss hidden gems that are essential
for mastering western blotting skills.
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