I performed an IP to pull-down an endogenous protein A. When I immunoblotted with anti-protein A, the film only showed bands for the input lanes. The first chemiluminescence treatment was with Pierce ECL, but no bands showed after 30 min exposure time. Then I used Pico Super Signal and got strong bands for the input lanes. If input is only ~ 1/10th of the total lysate, why did my IP lanes come out blank?
The protocol and reagents worked last week, but I have to prove repeatability. I plan to strip and reprobe, any suggestions on what might be going on with this experiment?
I appreciate your time and thoughts! Thanks!
Hi Kanomi. If you are not seeing the whole gel, at least in outline, after 30 min exposure with ECL then there is something wrong.
Did you do Ponceau staining of the membrane to see if there is anything there? Is there any sodium azide in your blocker?
Hi Kanomi, if IP actually worked you should see aproximately 10 times input in your eluate. Perhaps youre losing your protein with washing. High salt concentration or detergent presence may cause this kind of issues. Whats exactly the protocol?
Thank you all for your help. I am new to protein analysis and appreciate all the assistance I can get.
I did not do a Ponceau stain, but since the input was there I assumed the rest should have been there. No sodium azide in blocker.
I do three washes with my lysis buffer which has 150mM NaCl, but i do not think it is the washes. I think, either the agarose beads are not binding to my protein or they are not being eluted off. I think this because the incubation with beads is the only difference between input and pull-down samples.
I have made another lysate and will use another protocol which uses hypotonic buffer before lysis buffer. Any suggestions?
Hi Kanomi, are you using the same antibody for IP and probing the blots?
If the Ab is very good (or a poly) it can stay on the protein and block further Ab binding. Boiling samples in SDS-loading buffer with BME before running should fix it.
150mM salt wouldn't elute your protein, you're right. So, how do you elute your protein from the resin? As Steingrimur said, you can check if your protein keeps there with denaturing conditions. If you think that resin is not binding your protein, run the percolate in a lane. The protein can't disappear!
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