immunoprecipitation problem - (Feb/08/2006 )

Hi!!

I have some problem with immunoprecipitation.... I'm styding Src kinases family, which has 60 KDa of molecular weight... the problem is: when I reveal immunoprecipitates with anti-Src antibody, a enormous band appears (IgG) which doesnt let me watch Src band because of both, src and IgG bands, are very close...
What can I do to avoid this fact???
Thank you!!!

Hi-


You can try one of several things. You can really limit the amount of antibody you use and can run out the gel extremely far and play with the gel percentage to get the maximum seperation possible.

Another option is a secondary western antibody from eBioscience called Trueblot, which supposedly doesn't bind denatured IgG and hence doesn't bind your Ab in the gel, only the western detection antibody. Someone in our lab tried it with semi-decent results. I think some other companies are also starting to try to develop other products to address your problem as well since it is pretty common. I seem to remember Santa Cruz for some reason...you might try their website too.

Another option is covalent coupling of the antibody to protein G/A beads. There are several protocols for this, one of which uses dimethyl pimilimidate. You might try googling it to find a protocol, but if not I can send you one.


Good Luck,

Mountainman

Hi varince and mountainman,

I have the same probelm too. My target protein with the size 55 kDa, is almost overlapped by the IgG band. Therefore, I would like to try the cross-linkage protocol to cross-link the antibody with the protein A/G.

Mountainman, although I dig out some protocol from google, could you also send me yours for more ref.? Thank you very much.

Also, I have seen someone will run the western blot in the non-reducing condition, i.e. No DTT or BME in loading buffer. Is it really help for removing 55 kDa IgG band??

HI-

I have tried running a gel without DTT, BME, but with SDS in hopes of not seperating the heavy and light Ab chains. It diminished my light chain bands, which were masking my 25kDa protein, but not completely.


For coupling I use DMP (dimethyl pimelimidate). Be sure to pH the solutions carefully, because the idea is the DMP is alpha amine reactive and not (or less) for instance epsilon amine reactive at these pH's.

-Mix appropriate amount Protein A/G beads and antibody in 10 volumes lysis buffer
-Rock 1 hour at room temperature
-Wash the beads with 10 volumes 0.2M NaBorate (pH=9.0) by centrifugation at 3000g for 5 mins or 10000 for 30s
-Resuspend beads in 10 volumes same borate solution and remove a bit of the beads (for later quality control)
-Add enough DMP (solid) to bring final concentration to 20 mM
-Incubate at RT for 30 mins; remove a bit (like 10ul) of beads for later QC)
-Stop Rxn by washing once in 0.2M Ethanolamine pH=8.0 (again by spinning)
-Then add more Ethanolamine and incubate in it 2 hours at RT with rocking
-Spin and resuspend beads in PBS + a bit of azide (keep in fridge till use(they keep a while))

I would recommend running your beads before and after coupling on denaturing SDS-PAGE and blotting with secondary to see if coupling is complete the first couple times you try this. You shouldn't see any heavy chain band in the after lanes. Although there is sometimes still a bit of light chain though in my hands.

Good Luck,

Mountainman

Thank you very much for your answers!!!
I'm trying your advices!!!

QUOTE (Nagroc @ Feb 10 2006, 02:25 AM)

Another option is to do the westernblot with an antibody made in another specie different from the specie in which the antibody for the immunoprecipitation is made. In other words, if you make the IP with a polyclonal antibody (usually rabbit), then use a monoclonal antibody for the westernblot detection (usually mouse). The monoclonal antibody doesn't detect the rabbit immunoglobulins.

Hi Nagroc,

I have done what you suggested. IP protein (A) with rabbit polyclonal antibody, and detect protein ( with mouse monoclonal antibody. However, the heavy chain band still appear. I think it is because of the cross-reactivity (althogh it is low) of the secondary antibody to the IP antibody. It is the case. How can we deal with it?

Hi-


Interesting that you ask about the Borate Solution. I used Na Tetraborate decahydrate once and had problems with precipitation. Then I tried Boric acid, which didn't precipitate but, obviously isn't NaBorate, and the coupling still worked. I think you could honestly use any non-amine containing buffer in the pH8-10 range and it would work.

Good Luck,
Mountainman