How to stain proteins in a PAGE gel with Coomassie Blue

2015-09-30

If you're looking to excise a band from a PAGE gel after using Immunoprecipitation to purify a protein, staining all the protein in the gel can be useful. In steps the Coomassie Brilliant Blue dye. Originally developed for textiles, it's now routinely used to stain protein. There are many protocols out there for this process, but the one we find works best is below. It's also compatible with downstream mass spectrometry.

1. Fix PAGE gel for 30 minutes in 7% Acetic Acid 40% Methanol solution
2. Drain fixative and stain gel in 4 parts Coomassie G-250 colloidal concentrate (Sigma B2025) 1 part Methanol for 4 hours – over-night (~10 mL per mini gel is sufficient)
3. Destain gel in 10% Acetic Acid 25% Methanol solution for approximately 1 hour
4. Rinse with 25% Methanol to remove the Acetic Acid
5. Destain for up to 24 hours with 25% Methanol, or until bands are clearly visible
6. Proceed with imaging or excision of bands