Problem in IP (too high background) - mAb shows a lot of backgroud+light chain. (Apr/01/2007 )

:angry2:mAb shows a lot of backgroud+light chain.

Hi folks,

Recently I have a problem in IP. The purpose of this experiment is to find some interactors with a kinase (say, 'K', MW=36kD) of my interest and do Mass Spec. So first of all I did IP for 'K' with mouse monoclonal Ab or rabbit monoclonal Ab and tried to do western blotting. Below is some reagents that I used;

cell lysis buffer (protocol from KINEXUS, www.kinexus.ca):
-20mM MOPS (pH7)
-2mM EGTA
-5mM EDTA
I added protein phosphatase innhibitors cuz I am working with 'kinase';
-30mM NaF/40mM b-glycerophosphate (pH7.2)/20mM Na-pyrophosphate/1mM Na orthovanadate/
-protease inhibitor cocktail (Roche)
-0.5% NP-40

==> set to pH7.2

After cell lysis, I used 1.25 mg lysate each and precleared it for 2hrs with 50ul of washed bead (santacruz). Then transfered supernatant and added 5ug of anti-K antibody (either mouse/rabbit) including ctrl IgG, incubated O/N at 4C. Next day added 50ul of bead again and incuabed for 2hr and then remove supernatant and washed bead 5 times (x 5min) with same lysis buffer using rocking platform at 4C. Then pelleted beads and removed supernatant and added 50ul of 2x LDS sample buffer (w/200mM DTT in water) (Invitrogen, NP0007) diluted with lysis buffer. Then boiled for 5 min. Finally loaded into 8% SDS PAGel (Biorad).

Transfered to membrane O/N at 32V and next day blocked the membrane with 5% milk and incubated with anti-K mouse monoclonal Ab for 4hrs at Room temp. Then washed, incubated with secondary anti-mouse, washed and blotted.

The problem I have is that the monoclonal antibody works always good for regular western blotting but I am not sure whether this one and rabbit one are good for IP or not although the product information sheet says I can use both for IP and for western blotting. In addition, although two left positive ctrls looks good (input) but it is hard to see both IP'ed wild type K and mutant K on the right. Actually the picture that I uploaded is IP with rabbit antibody and blot with mouse antibody (mouse antibody IP-mouse antibody blot looks more ugly).

Is there anyone who knows how to solve this problem?

Thanks in advance!!!

anti-mouse cross-react with rabbit Ig and vice-versa.
there are several ways to solve your problem :

- detect with protein A-HRP instead of anti-mouse or anti-rabbit. Protein A recognizes mainly the native immunoglobulin and not the denatured one.
- or use CnBr activated beads, to cross-link the antibody for IP to the beads.
- or biotinylate the antibody used for western-blot and use streptavidin-HRP for detection.

Can that be the ammount of antibody for IP which induces the problem? I mean too much antibody (5ug) which can be washed off by sample buffer and be detacted when you do western.. I guess.

the problem in this protocol is that you load also your antibodies for IP on the gel, and when you detect the primary antibody with the anti-IgG-HRP, you also detect the denatured Ig that were used to IP and that migrated and transfered to the membrane.
even if you would reduce the amount of Ig for IP you would detect them.