10 Tips For Western Blot Detection Of Phosphorylation Events

The ability to interrogate protein phosphorylation events and aberrant phosphorylation levels is critical to our understanding of various biological and pathological processes, particularly in the research of diseases, like cancer. However, detection of phosphorylated proteins in western blots can be challenging. We have developed ten tips to ensure robust data generation and cleaner blots.

Topics include:

1. Inhibition of protein degradation and conservation of posttranslational modifications during sample preparation
2. Selection of phospho-specific antibodies
3. Probing for total protein
4. Induction of phosphorylation
5. Treatment with phosphatase
6. Concentration of samples
7. Optimization of blocking reagents
8. Types of membranes
9. Washing
10. Analysis of total protein normalization (TPN)