Hi all, I am trying to detect p-IRF3, p-STAT1 and p-STAT3 proteins.
When I used 5% milk with TBST to block my membrane and dilute my primary and secondary anti-phospho antibodies, I got back a blank membrane as seen in the picture. I then switched to using 5% BSA in TBST to block my membrane for 1h at RT, and diluted my primary and secondary anti-phospho antibodies in 3% BSA with TBST. However, I got back a lot of non-specific binding, to the point where I am unsure whether my target protein is even present (as seen in the picture). Any tips? Should I continue using BSA or should I switch back to using milk?
Also, I noticed that after switching to BSA, my pre-stained protein ladder fades completely after blocking and washing i.e the transfer was perfect, but after blocking and washing, the protein ladder disappeared. What should I do? Because I wont be able to interpret results without a protein ladder.
FYI:
Primary antibody dilution is 1:1000
Secondary antibody dilution is 1:5000
Thank you all for your help!
Hello Maddie Yeo
It is better to use BSA. Milk should never be used 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.
Decrease your secondary antibody concentration. Dilution of 1:5000 is too high for secondary. Use a dilution of 1: 15000 or 1: 20000 dilution for the secondary antibody. This will reduce the non-specific bands. You can also increase your blocking to 1.5 hrs if required.
Use less stringent methods for washing. Using high washing speed could wash away the protein ladder. So, you can turn to low-speed washing. This could retain your pre-stained ladder.
Also, you need to add Phosphatase inhibitors like sodium orthovanadate and sodium fluoride in the lysis buffer to prevent dephosphorylation of phosphorylated proteins.
I hope this helps.
Good Luck.
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