non-specific binding to anti-Flag beads - (Apr/20/2006 )
I am trying to perform an experiment that tests whether a short N-terminal del mutant of my protein can multimerize with the wild type. (WT multimerizes). My del mutant is tagged w/ HA and my WT w/ Flag. I IP'd with anti-Flag beads (overnight, rotating at 4 C), then washed x3, boiled X5 min in Laemmli buffer and ran on 12% gel using untransfected cells and IP ingredients as neg controls. On my blot I have an unique band in the cotransfected sample (yay!) but also one in the del mutant alone. Is it possible that my protein or even the HA could have bound non-specifically to the anti-flag? The band is the right size...
First of all I would like to ask you a few things:
- Did you co-transfect cells with WT-HA, WT -FLAG ?
If you didn't you should as it is a good positive control. You know WT protein forms multimeres so IP should work.
You can IP with FLAG come back to HA to see if HA-WT was IP.
- You Ip with FLAG beads right? Did you come back with FLAG antibody to check wether you sucessfully IP FLAG WT protein. If you see you didn't IP then you know the probelm is the IP. If you could IP, then you can conclude that in the conditions tested the mutant protein didn´t interact with WT protein.
- Which lysis conditions have you used? Maybe you are using too much detergent, that can disrupt a real but weak interaction?
yes, it is possible that your protein binds non-specifically to the beads.
it is good idea to first optimaze the IP conditions using two, differently tagged WT proteins - so FLAG IP of HA-wt alone should be clean, and you detect interaction only when cotransfect with Flag-wt. then you incude your mutant, and you should get your answer.
Yes you should optimize IP conditions.
Why don't you incubate lysate with antibody for 1- 3 hours instead of ON ?
You might increase binding specificity?
What you can also do is to pre-incubate lysate with beads only for 30min transfer the lysate to a fresh tube and incubate again with the beads for the desired time 1- hours or ON. This extra step is called pre-clearing of lysate. This way you eliminate unspecific binding of proteins that bind to protein A or protein G, independently of the presence of the Ab.
Thanks for your help - I didn't mention it but I do preclear for 1 hr. I now have a WT for each tag and will run individually. I also will try the shorter incubation time. That is a really good idea!