Dear Caleb,

The following paper covers the answer to your question. You might need to get the full paper.

Protocol

Nature Protocols 2, 3278 - 3284 (2007)

Published online: 13 December 2007 | doi:10.1038/nprot.2007.459

 http://www.nature.com/nprot/journal/v2/n12/full/nprot.2007.459.html

Subject Category: Isolation, purification and separation

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Yuliang Wu

Qiang Li

Xing-Zhen Chen

Detecting protein–protein interactions by far western blotting

Yuliang Wu1,2, Qiang Li1 & Xing-Zhen Chen1

Abstract

Far western blotting (WB) was derived from the standard WB method to detect protein–protein interactions in vitro. In Far WB, proteins in a cell lysate containing prey proteins are firstly separated by SDS or native PAGE, and transferred to a membrane, as in a standard WB. The proteins in the membrane are then denatured and renatured. The membrane is then blocked and probed, usually with purified bait protein(s). The bait proteins are detected on spots in the membrane where a prey protein is located if the bait proteins and the prey protein together form a complex. Compared with other biochemical binding assays, Far WB allows prey proteins to be endogenously expressed without purification. Unlike most methods using cell lysates (e.g., co-immunoprecipitation (co-IP)) or living cells (e.g., fluorescent resonance energy transfer (FRET)), Far WB determines whether two proteins bind to each other directly. Furthermore, in cases where they bind to each other indirectly, Far WB allows the examination of candidate protein(s) that form a complex between them. Typically, 2–3 d are required to carry out the experiment.

Hoping this will be helpful,

Rafik

Try to wash the membrane with 0.1% TBST overnight. Good luck!

I think you can use vacuum pump for blotting the un denaturated protein but with high purity to avoid clogging of the membrane. reacting with monoclonal antibody will determine either its the right protein with its right epitope conformation.