HelP---antibody does not work - ChIP (Jul/17/2005 )
Hi guys, I have wasted more than two moths on the immunoprecipitation step but made no progress.
I'm working on a plant protein. Here is the binding condition that I used, it works well for several other plant transcription factors:
chromatin pellet was resusended in 1X sonication buffer, after sonication, the supernatant was taken and mixed with an equal volume of 1X IP buffer:
sonication buffer: 10mM phosphate buffer, pH 7.5, 0.1M NaCl, 0.5% sarkosyl, 10mM EDTA, with freshly added PMSF (1mM) and protease inhibitor (Roche);
IP buffer: 50mM Hepes, pH 7.5, 150mm KCl, 5mM MgCl2, 10 uM ZnSO4, 1% triton x-100, and 0.05% SDS.
The primary antibody was generated from goat (santa cruz), I used G-Plus agarose beads (also from santa cruz) as matrix. primary antibody was added first and incubated for 2 h, followed by adding G-Plus beads and incubation for 1 h. All steps were did at 4C.
From my western blot result, I was confident that my protein of interest was present in the solution before immunoprecipitation step, but it did not bind with my primary antibody, since it was found in post-bind solution (supernatant after G-Plus incubation) only. Besides, in the elute, I only found heavy chain and light chain of primary antibody. Furthermore, I think that protein degrade very easily, because the band intensity in post-bind has been greatly reduced.
I actually tested two different antibodies, one recognizes epitope on N-terminal region and the other recongnizes C-terminal. Both of them gave me similar results.
by the way, I did a lighter fixation compared to other plant ChIP protocols, therefore, chance of epitopes been damaged would be quite low.
I think I really really need some suggestions from you. what's wrong with my IP process?
Thank you very much!
I am not very familiar with plants, but it may help to IP for a longer period of time, I usually go overnight. I also incubate with beads for 1-3 hours. Maybe add different protease inhibitors, or make sure they are not diluted less than working concentration when IP buffer is added. Hope this helps!
We got gift from another lab, they used serum instead of commertialized antibody and the epitope they chose is the middle region --- it works!!!! I didn't change binding condition at all!! hi beccaf22, thanks for your advice, as you suggested, I will do a longer period of incubation because only around half of the protein was captured this time.
In my lastest Western blot, I also loaded protein sample without boiling (I just added loading dye and mix), no signal was found; whereas the boiled sample (adding loading dye and 95C for 5min) gave clear bands at correct MW.
The protein structure should be the reason.