I am trying to detect an endogenous protein of around 135KDa by Coomassie or Silver Stain for a downstream analysis. My immunoprecipitation works well, and the protein seems highly enriched in Western Blot, but I have so far been unable to detect it by staining the SDS gel directly even after pooling multiple lPs. Any suggestion would be highly appreciated.
Both stainings should have detected immunoprecipitated protein. Try preparing Coomassie like this:
50% Methanol, 10% Acetic Acid with 1g of Coomassie Brilliant Blue.
Stain for 30 min.
Unstained with 20% Methanol and 10% Acetic Acid for 2h and add cellulose (paper) with your gel to bind excess stain.
Thanks Mateo, I will try it. My silver staining revealed numerous bands (including around expected size), but no clear distinction between the IP lane and IgG control lane. That shouldnt be the case yea?
Western is the best way to detect this. Coomassie is not sensitive enough. Silver could work, but if your IP is dirty, your band can be obscured. If your Western is working, why do you want to detect it with a non-specific method? This ought not to be necessary, unless your situation is somehow weird.
Ayon Hassan
No, you should see the immunoprecipitated protein as the strongest bend except for heavy and light chains coming from the antibody (which should be the same in IgG control). I usually see the precipitated protein after staining the nitrocellulose membrane with Ponceau S. Maybe the endogenous expression of your target protein is too low. Try changing the cell line.
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