I repeated my western blot 5 times with 5 different samples, trying to detect phospho-IRF3, phospho-STAT1 and phospho-STAT3. Sadly, all 5 of them had totally no bands present for all 3 proteins (but GAPDH signal was really strong, so I know my proteins have transferred). Any tips?
I use 5% milk to block my membrane for 30mins at room temperature, and 5% milk to dilute my phospho antibodies and incubate them overnight at 4°C. Will it be better to use BSA? And if so, how do I prepare them with BSA?
Hello,
Yes, it is better to use BSA.
You should never use milk while detecting phospho protein by Western Blot. Milk contains casein, a phosphoprotein that will bind to anti-phospho antibodies causing non-specific binding and high background. Milk may mask some antigens if they are present in low abundance which would result in faint bands.
For detection of phospho protein by Western Blot you should use 5% BSA in TBST for blocking, primary antibody in 3% BSA in TBST and secondary antibody in 3% BSA in TBST.
Also, you need to add Phosphatase inhibitors like sodium orthovanadate and sodium fluoride in the lysis buffer to prevent dephosphorylation of phosphorylated proteins.
Best Wishes.
Malcolm Nobre alright, thank you! just to confirm, I just dilute my BSA in TBST?
Hi. Are you using phosphatase inhibitors in your cell lysis buffer?
Another reason could be your time points or treatments for these phosphorylated proteins. You could include positive controls for your phospho-proteins.
5% BSA/1xTBS-T is usually recommended for phospho-protein detection with WB analysis.
Enkhtuya Radnaa Yes, I added 1 tablet of phosphatase and protease inhibitor to 10ml of RIPA buffer for cell lysis. Not sure if that is sufficient. I'll add a positive control the next time, thank you!
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