- Proteins and Peptides
- Lysates and Cell Lines
By Jamshed Arslan, Pharm. D., PhD.
Protein phosphorylation refers to a reversible post-translational modification in which a protein kinase adds a phosphate group to an amino acid residue of a target protein. Protein phosphorylation, especially tyrosine phosphorylation, is one of the early events in signal transduction in all eukaryotic cells.
Once a cell is lysed, proteases and phosphatases are released that can degrade or modify proteins, thereby affecting their Western blot detection. The following are some tips for improving Western blot analysis of phosphorylated proteins.
1. Keep samples on ice and use pre-chilled buffers
2. Use phosphatase inhibitors
3. Store samples in the loading buffer
4. Avoid milk as a blocking agent
5. Use phosphate-free buffers
6. Use sensitive substrates for chemiluminescence detection
7. Detect total protein content
Independent Antibodies Validation and Biological Strategies Validation. AKT1 [p Ser473] Antibody (104A282) [NB100-56749] - Mouse 3T3 cells were treated with and without PDGF (50 ng/mL) for 0 to 60 minutes. Total protein from whole-cell lysates was separated on a 7.5% gel by SDS-PAGE, transferred to PVDF and blocked in 5% non-fat milk in TBST. The membrane was probed with 2 ug/mL anti-AKT1 (NBP2-01725) and 2 ug/mL pS473 AKT1 in 1% BSA/ TBST and with an anti-mouse HRP secondary antibody. Signal was visualized with chemiluminescence detection.
To quantify a phosphorylated protein, fluorescent conjugated antibodies can be used to detect phosphorylated and total proteins on the same blot in a process called multiplexing. Fluorescent tagged secondary antibodies generated in two different species can show one color for the phosphorylated form and another for the total protein.
In conclusion, WB of phosphorylated proteins is essentially similar to the non-phosphorylated ones, but caution must be exercised to avoid dephosphorylation during sample preparation. The above-mentioned tips are easy to follow and can significantly improve phosphorylated protein detection through Western blot.
Jamshed Arslan, Pharm D, PhD
Dr. Arslan is an Assistant Professor at Dow University of Health Sciences, Pakistan,
where he teaches Pharmacology to future pharmacists.
Bass, J. J., Wilkinson, D. J., Rankin, D., Phillips, B. E., Szewczyk, N. J., Smith, K., & Atherton, P. J. (2017). An overview of technical considerations for Western blotting applications to physiological research. Scandinavian Journal of Medicine and Science in Sports. https://doi.org/10.1111/sms.12702
McDonough, A. A., Veiras, L. C., Minas, J. N., & Ralph, D. L. (2015). Considerations when quantitating protein abundance by immunoblot. American Journal of Physiology - Cell Physiology. https://doi.org/10.1152/ajpcell.00400.2014
Murphy, R. M., & Lamb, G. D. (2013). Important considerations for protein analyses using antibody based techniques: Down-sizing Western blotting up-sizes outcomes. Journal of Physiology. https://doi.org/10.1113/jphysiol.2013.263251
Sawasdikosol, S. (2010). Detecting tyrosine-phosphorylated proteins by western blot analysis. Current Protocols in Immunology. https://doi.org/10.1002/0471142735.im1103s03