how to determine protein-protein interaction - (Apr/07/2007 )

We got a novel secreted protein, but I don't know how to test the interaction of this protein with its potential partner. Two-hybrid system or pull down is only for intracellular scenario. Am I right? Hopefully, anybody can help me address this question.

hi,

surface plasmon resonance
no need to label any of your proteins, just chemically link one of the two protein to the chip and pass the second
think about a control protein to put on the second flow cell

Sebastien

Maybe I need read some documentation to get familiar with that. Anyway, thanks a lot.

If your protein is a GST fusion, you could try a GST pulldown. Take purified GST fusion, and mix it with a lysate of the cell of interest containing the putative partner protein. Following an incubation step, pass the lysate over a fresh glutathione column. Wash carefully, but not too harshly; your target protein should remain bound to the fusion protein, which will be stuck to the column. Elute the complex with glutathione, and run the eluate (along with load, and a few washes) and see what comes up!

Thank you very much for a good suggestion, swanny!

hi

can u give me a details of this surface plasmon resonance method? i would be grateful.
thx