Problems with Immunoprecipitation and Cross-linking

[kiki06]

Hi,
I am conducting IP on a 70kDa factor. I am trying to do IP and pull down with
Dynabeads -Protein G from Invitrogen. It is not working at all. Does anyone have any experiences with pulls downs with IP then western blot / detection?

How can i be sure the crosslinking worked?

thanks

[danfive]

Are you crosslinking your Ab to the bead? If so you can test that by quenching the reaction (usually Tris buffer), washing, eluting (perform usual elution step), equilibrating then doing a Protein Quant assay. If you have protein means your Ab eluted and crosslinking wasn't 100% effective. If no protein is detected by Protein Quant Assay your Ab is linked to the beads.

[kevin_a]

Kiki,

Macroboy had similar problems with the experiment not working.
To test the crosslinking he just ran the elution fractions on gel and did the Western looking for heavy chain. (not many people are willing to go through that much trouble)

However, since your protein is 70K, you could probably get away without doing the crosslinking. Especially if you have a monoclonal antibody. Sometimes they do not take to kindly to being crosslinked.

K

[marcroboy]

Hi Kiki,
I guess if I checked this forum more often I could've replied earlier, I'm not sure if this is still troubling you.
Anyways, like kevin_a said, I was trying to do a similar pull-down experiment with dynabeads protein G. My cross linking reaction did work except that I couldn't get method of eluting just the protein. I tried Citrate ph 3 buffer in their manual which gave me no protein, however when I boiled the same beads in SDS loading buffer I saw the protein on the western blot gel.


However, I ended up forsaking the cross-linking because like Kevin said,if you can resolve the protein from the antibody heavy chain, you shouldn't need cross-linking. And in my case, I had a negative control antibody(preinjection serum) which I ran along that clearly told me that the additional band is the protein that I pulled down.

[marcroboy]

Oh also just in case anybody else is watching this thread, does anyone reuse these dynabeads, I tried to reuse the beads I did IP with earlier(they were boiled in loading buffer) and the resut didn't come very good. The manual says they could be reused for up to 5 times but I guess it depends on the method of elution? These beads are pretty expensive so I don't really feel like wasting them because for my next procedure I plan to do a lot of IPs.

And another question I have regarding the storing of Protein G-Ig complex. For all of the IP I ran, I used fresh immunoprecipitated products but I am wondering if protein G-Ig complex can be stored in say the washing buffer in -4 for later use(to capture my protein), would Ig degrade or dissociate over time? I wanted perform Casting on it which requires multiple cycles of IP hence if I can make a whole bunch of protein G-Ig complex in one batch, it would save me a lot of time in the long run.