IP problem: antibody binds too few antigen - (Feb/19/2009 )

Thanks for all the answers.
This sterical inhibition is something to consider. Next week I'm going to try IP with more beads (I used 20 mcl of it (50% packe volume). I'll go on with 40 mcl. And anyway I will try the "antibody first with the bads" method too.
But unfortunately I can't affors to use antibidies of different orgin to IP than to the subsequent Western blot.

Increasing the beads will do nothing but increase your background or non-specific binding. I am a strong advocate for prebinding the antibody to the bead before adding lysate. I recommend the following: take 10-15 ul of protein A agarose beads. Wash twice and add antibody with lysis buffer or pbs. Let bind for at least one hour at room temp with the tubes agitating end-over-end. It's important to keep the beads in slurry and not at the end of the tube. Wash the beads twice with buffer and block your beads in fresh made and filtered 5%BSA/PBS for at least another hour but I prefer four hours to overnight. Meanwhile, make your lysate and pre-clear by incubating with control rabbit IgG bound to agarose beads. This helps remove non-specific binding in the IP. After at least one hour but again, I prefer at least four, carefully remove the supernatent from the preclear beads (you don't want to add any of the non-specific binding back to the IP). Bradford the lysate to check protein concentation. Wash your BSA-blocked beads at least 2-3 times. It's critical you get out any free BSA. Add lysate, incubate for 4hours to overnight. The time needed here completely depends on the antibody. Longer times might increase the antigen IP but might also increase background. This you much optimize to your own conditions. Wash the beads well, boil in 20-30ul sample buffer and run on the gel. For detection in the western, because you are using both rabbit antibodies in the IP and the western, you are lighting up the heavy and light chains of the IP antibody which makes the western (especially for a 56kDa protein) nearly impossible. Rather than using anti-rabbit HRP as your secondary, you should try protein A-HRP. Otherwise I can't stress the importance of having a mouse antibody for the western blot. If money is an issue, find a group that published with the mouse and request an aliquot. Call the companies and beg for a sample aliquot. There are many ways to get your hands on antibodies without paying for them. Also, if your advisor has published a paper using a santa cruz antibody (and I think abcam has starting doing this as well) you are entitled to a FREE antibody!

leptopelis on Feb 19 2009, 03:58 PM said:

since you are using antibodies from rabbit i assume that they are polyclonal. were they made against a small portion of the protein or the whole molecule?

with polyclonals, especially if made against whole protein, you will see more than one molecule of antibody binding to one molecule of antigen.

you can see how this might affect the intensity of staining.

Hi again,

I think hormone receptors can be very difficult to IP.
You might want to think about transfecting in a myc-tagged estrogen receptor or FLAG-tagged and IPing
with the Flag (or myc) antibody.
Here is a recent paper where they did some co-IP with the Estrogen receptor.
Note that they use different species of antibody for IP and Western
However, if you are Co-IPing for the Estrogen Receptor and Western blotting for the Estrogen receptor the
intensity fo the band in your Western should be strong enough to out power any background.

Good luck.

Regulation of estrogenic effects by beclin 1 in breast cancer cells.
John S, Nayvelt I, Hsu HC, Yang P, Liu W, Das GM, Thomas T, Thomas TJ.
Cancer Res. 2008 Oct 1;68(19):7855-63.