Antibody not binding to protein A/G - Immunoprecipitation (Sep/25/2007 )
I am using a homemade polyclonal rabbit antibody with 1% BSA and NaAzide to immunoprecipitate my protein of interest. I am using Pierce's protein G kit and follow the instructions exactly. After incubation of the antibody with the immobilized protein G, all of my antibody comes off in the flow through (I can detect a band at 150kDa on a silver stain). I have tried protein A, longer incubations, and changing the pH from 7 to 5, but nothing has worked and my antibody just won't bind! Has anyone seen this before? Any advice? Thank you.
I have several questions:
- How do you know that your antibody was washed off? You run the flow through and stain? But if you prepare the sample (that you run on gel) with normal sample buffer (including either DTT or 2ME or such), then usually, we find heavy chain and light chain bands, not one band. Furthermore, you detected this band only after silver staining? Then this is a very small amount of Ab (assuming that this IS the Ab stain). How much Abs did you use in the starting material? Surely if you use a kit then you wouldn't start with too little Ab, right? Cannot be just 1-2 ug, right? If so, the band you observed should not be ALL your Ab, rather, just a very small amount.
- Maybe I am wrong, but wouldn't we normally use Protein A for polyclonal rabbit Ab, instead of protein G? Also, didn't low pH is one way to ELUTE Ab (as in, release it from binding)? According to the Pierce information, optimal binding pH for Protein A is 8.2, while for protein G is 5. If you use pH 7 for protein G and pH 5 for protein A, then.....
Check this out:
When I run the silver stain I have a postive control of stock Ab and they have relatively equivalent large bands at 150KDa. I do have 2ME in my sample buffer (I don't boil it), so I am not sure why the disulfide bonds aren't broken (maybe they are partially broken) and I don't see heavy and light chains. I started with about 120ug of purified Ab in the sample that I put on to protein A or G. For protein G, I tried the binding buffer at pH 7 and then at pH 5 incubating at room temp for 20 minutes. After this didn't work, I tried protein A at pH 7 incubating at 4C overnight. Do I have too much BSA in my sample? Do some rabbit Abs just not work for IP?
- I am not sure if it is because of boiling or not, but usually you should observe two bands for Ab. That asides, as I said before, is the amount of Ab in the flow through equal to the amount of starting Ab? You may got exceed Ab washed off in the flow through, but that is different from Ab not binding to protein A.
- According to the information page, which I linked to in the previous post, protein A or G does not bind BSA, so it should not be a problem
- Abs, regardless of the origin, may or may not work for IP, but it is not the problem here, because the issue is the binding of Ab with protein A or G. Did you run your beads after binding? Or do anything to check if the Ab has been bound with the beads? I am not familiar with the kit that you are using, but if that kit does not include cross-link Ab with beads, then when you run the beads you should got Ab there and you can determine if your Ab bind or not bind to the beads, and how effective is the binding.
- I also notice that the pH that you used is not the optimal pH for either protein A or G to bind to Ab. And why did you choose protein G as your first choice?