Originally Posted by kevin_a
Not sure why they recommend a pH 3. We suggest people avoid doing that because of the leaching. Your incubation time is short, so that may prevent significant leaching. Then again my intel could be wrong about the chemistry on the Dyna beads, but I do trust the source.
If you just want to check the crosslinking efficiency: Before you do an actual experiment, perform a Western on the washes and pre-elutionion fractions with anti-IgG after you do the cross-linking so you can see what's coming off and when. If you see IgG chains light up on the Western at 25, 50, 75 and 150 then - the crosslinking is not working. The recombinant Protein G is ~22 kDa, so bands additional bands at 22, 47, 72, 97, 172 would indicate the Protein G came off with the IgG.
There is also another possible explanation for why you don't see your protein of interest. The elution conditions may be too mild. More common than you might thins, some IgG will bind very tightly to antigen and then resist standard elution conditions. Your protein is 40 so you should be able to resolve that from the mess that will result by simply boiling in reducing SDS sample buffer after the IP and doing the Western.
If you need more detailed help, feel free to keep posting or email me.
I think I have the answer to my failed immunoprecipitation: as it turned out, my purified protein sample was degraded, probably because of the lack of protease inhibitor. So yea, no wonder I was having problems, I was IPing out of nothing
However, I have run an assortment of elutions under different conditions to test my cross-linking efficiency. I hope with your familiarity with this technique you could give me some pointers.
I ran an IP without
cross-linking then I eluted with 0.1 M citrate pH 3 buffer. As expected I see the heavy chain and light chain band under comassie (distinctly). Furthermore, with the same bead,I also boiled them under reducing loading buffer after the first elution, I could also see the antibody band(faintly). And this band is slightly higher than the acid eluted band.
On a different set of beads, I ran the IP with cross-linking. First elution with citrate pH 3 buffer gave me a faint heavy chain band. The second acid elution gave me nothing.
Consequently, I boiled the same beads under reducing loading buffer and again it gave me the heavy chain band. And just as the uncross-linked case, this band is slightly heavier than the acid eluted band.
I guess the second case would suggest that crosslinking worked? I didn't get antibody with the second acid wash however boiling them under reducing condition resulted in the antibodies.
Furthermore, it seemed that every time I boil the beads, the resulting band is always a bit higher than the acid eluting the beads. However, I can't imagine what else would be coming off besides the antibody and protein G. What are the chances that protein G are coming off with boiling?