help!! immunoprecipitation - (Aug/06/2009 )

I have tried several times on the immuprecipitation. But I only can detect the heavy chain but not my target protein. The antibody I used for the IP was published by a few groups who used that for successful pull down of the target protein. The procedure of the immunoprecipitation which I did was that first I lysised the protein in lysis buffer with proteinase inhibitors ( I tried several different buffers from non-stringent to stringent, and PH from 7.4-8.0), and mixed about 500ug protein mixture with 2ug monoclonal Ab ON at 4C, and then added protein G beads around 40ul for 4hrs at 4C, I even didnot do the washing step, and finally eluted in SDS-loading buffer. But As I mentioned at the beginning, I could only detect the heavy chain in the final WB, but not my target protein ( The target protein is surely not covered by heavy chain.) Could u give me any suggestions on how to resolve this problem?

My lab sees this off/on all the time. Sometimes we correct for this by titrating back the antibody or the amount of resin. If there is too much antibody for the amount of resin (and obviously in surplus of the protein), you often get heavy chain bands. We don't worry about it if we can see what we are looking for, but occasionally it is problem because our protein run close the heavy chain. Sometimes the heavy chain signal sort of eclipes our bands.

You might also try changing incubation times as well. Typically, we bind the ab/protein for 30 min to an hour at 4 degrees before adding the resin. We continue binding for 2 - 4 hours at 4 degrees before washing and loading on the gel. I have bound overnight and for only an hour depending on the antibody and/or protein. I think you will just have to play with it a bit to see if you can get signal with minimum amounts of HC "background".

When you say you have tried high and low stringency, what are you referring to, the binding buffer or the wash buffer? You should try different stringencies for both the binding buffer and the wash buffer. If they are one and the same then try changing one or the other.

I haven't used it, but a neighboring lab use to use True Blot for the ECL Westerns. It is supposed to eliminate the HC and LC bands that sometimes interfere. I thought Amershan used to sell it, but I found it at the posted link. True Blot

Happy developing! http://www.protocol-online.org/forums/styl...icons/icon1.gif

How large is your protein? Is it a similar size to the IgGs?
Is the protein expressed in the cell type you are using?
Have you used a cell line that was previously used successfully by other groups?

Hi

Are you using Dynabeads for the IP? Have you tried both the direct and indirect method for IP?

When using Dynabeads Protein A or Protein G, one typically does not need to incubate for more than 10 min at room temperature, both for Ig binding and for beads-Ig-target coupling.

Do you have a positive control on your western blot? Perhaps the IP is working but the detection isn't?

Regards

Kristina

Are you running your lysate on the blot next to the IP samples? Can you see the signal in this lane? This will indicate if your protein is expressed and if your primary antibody (for your western is working)

In addition to needing an input as stated above, what species are your antibodies?
If the Ip antibody is rabbit you should use a different species for the Western?
Do you have proper controls? Have you done the IP using a cell line that was previously published?