coupling surface biotinylation & co-immunoprecipitation? - (May/22/2007 )

Hello,

First of all, sorry if the answer to my question has already been given in these forums... I was unable to find this information despite some time spent searching.

I am working with transfected cell lines. From the proteins expressed in my transfected cells, different combination exist on the cell surface but only one of those are of particular interest for me.

I don't think that, in these conditions, surface biotinylation would allow me to detect the surface expression of a particular combination.

However, I was wondering if somebody could tell me if coupling surface biotinylation and co-immunoprecipitation was feasible. Besides, if it is possible, I believe I'd have to uncouple biotin and avidin, which could require harsh elution conditions that may impair protein-protein interactions...

Many thanks for your help

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If you have antibody special for the combinations, simple surface biotinylation would allow you to detect it (just biotinylated and the streptaviden-beads to pull down, WB to detect).

If not, then of course you can do surface biotinylation and IP together (but doesn't IP imply that you already have Ab?). You can do either way: 1) biotinylated-lysed-IP-WB with streptavidin HRP or 2) biotinylated-lysed-Streptaviden beads pull down- elute (easiest to use NHS-s-s-biotin and use DTT to reduce the s-s bond and release the protein or to boil in SDS sample buffer without dye and 2ME or DTT)-IP-WB.

the 2nd method got advantage that you will get rid of the biotin in the protein (which could, although not often, affect the epitope and affect IP or WB) but DTT or boiling could affect your proteins, too. This method, without the elution and IP step is the same as just biotinylation and then detect surface protein using specific Ab.

Also, as for uncoupling biotin and avidin, it is not necessary. As I said, use NHS-ss-biotin (Pierce) to do biotinylation, bind to beads then reduce the s-s bond. You will got protein in the supernatant. The problem is, if your pro-pro interaction is also based on reducible bonds, then......

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Hi

Actually, yes, I have the good antibodies.
But when I express 3 proteins in my cells, A, B and C, A+B goes to the surface, as well as A+C while I am interested in A+B+C.
Having specific antibodies for every proteins is, I believe, no use in this case.

Having said that, I have found a solution to my problem in a paper in which they did:
1) biotinylation reaction
2) co-ip
3) elute the proteins from proteins G/A (with highly concentrated SDS)
4) pull down with Streptavidin

I still have to improve my surface biotinylation conditions but, overall, this protocol seems to work

many thanks

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Here's another solution.

Use the Pierce kit for Cell Surface Purification - it ends with releasing the purified proteins from the avidin resin by cutting off the biotin. This gives you free protein in concentrated form to do your co-IP with.

http://www.piercenet.com/Products/Browse.c...;WT.mc_id=forum

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