How to Use Western Blot for Detecting Post-Translational Modifications (PTMs)

Western blotting is an effective method for detecting post-translational modifications (PTMs) such as phosphorylation, glycosylation, and acetylation, which play a critical role in protein function. Here is how to analyze PTMs in a protein sample using western blotting.

1. Sample preparation

Prepare protein samples by lysing cells or tissues in a buffer containing protease and phosphatase inhibitors to prevent degradation of PTMs. Quantify protein concentration to ensure equal loading, which is critical for comparing PTM levels between samples.

2. Gel electrophoresis

Load equal amounts of protein (e.g., 20-50 μg per lane) onto a polyacrylamide gel and separate by SDS-PAGE. For some PTMs (e.g., glycosylation), you may need specific gel types or conditions (e.g., using gradient gels) to improve separation.

3. Protein transfer

Transfer the protein to a PVDF or nitrocellulose membrane to make it accessible to antibodies.

4. Blocking

Block the membrane with blocking buffer (BSA is preferred for PTMs, as milk-based blockers can mask some epitopes).

5. Primary antibody incubation

Modifications are detected using a PTM-specific primary antibody (e.g., anti-phosphorylation, anti-acetylation). These antibodies are often highly specific for the modified residue, so choose one that has been validated for Western blot. For comparison, total protein levels are also stained with an antibody against the unmodified protein to normalize for PTM expression.

6. Secondary antibodies and detection

Enzyme-linked secondary antibodies are applied to bind to the primary antibody, followed by visualization using a substrate.

7. Quantification

Modified protein band intensities are compared to total protein bands using imaging software. Normalizing PTM levels to total protein allows for accurate comparisons between samples.

PTM analysis using Western blot can provide insight into protein function and signaling pathways under a variety of conditions.

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References

[1] Juliane Hermann et al., Molecular Aspects of Medicine 2022 (https://doi.org/10.1016/j.mam.2022.101066)

[2] Alisa Woods et al., J Mol Psychiatr 2013 (DOI: 10.1186/2049-9256-1-6)