Band sizes after co-IP - (Sep/25/2007 )

Dear all,

I have a stupid question...

I want to show the interaction of A (size~52 kD) and B (size~55kD) proteins, after I did IP with the antibody against A protein, I did the Western blot with Anti-B antibody.
If A and B really interact with each other, should I expect to see a band around 107 kD on my western blot?? Or should the band still be 55 kD because the proteins were denatured??
If I see a 107 kD band, could it because of the cross-linker I used before IP??

Thanks a lot in advance!

Elaine

-Elaine Chou-

QUOTE (Elaine Chou @ Sep 25 2007, 01:16 PM)

If you crosslinked, then yes but it would depend on the method of crosslinking (formaline, UV, etc) since they each have their own "reach" at the molecular level (as well as substrate of course). Otherwise.. in a denaturing SDS-PAGE gel... they should be at or near their native MW.

-jonathanjacobs-

Dear Jonathan,

Thank you for your reply!
The crosslinking reagent I used is DTSSP (3,3'-Dithiobis[sulfosuccinimidylpropionate]) from Pierce. Have you ever used this crosslinker? Do you have any idea if this reagent may cause the size difference?

Thanks!!

QUOTE (jonathanjacobs @ Sep 26 2007, 10:56 AM)

QUOTE (Elaine Chou @ Sep 25 2007, 01:16 PM)

-Elaine Chou-

Nope. I've never used it, sorry. But, from a quick google search and a review of the product page I would say that it _could_ be the source of your mystery band if SDS-PAGE buffer lacked BME or you did not reverse the crosslinks. Also, you may need to confirm this for your set up, but I've seen heavy- and light-chain dimers in my gels before from an IP if my boiling was incomplete which can be ~100Kd

http://www.piercenet.com/Products/Browse.cfm?fldID=02030244

-jonathanjacobs-

Hi Jonathan,

Thanks for your kindly reply!
I guess I didn't add enough b-ME. I used 1% and they suggest 5%...
I'll try to add more and see what happens!

Thanks again!

Elaine

QUOTE (jonathanjacobs @ Sep 26 2007, 05:41 PM)

-Elaine Chou-