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GST pulldown optimization - (Nov/13/2007 )

Hi, I´m performing GST Pulldown to identify protein-protein interactions between GST-X and prey proteins in a cellular lysate. The results are not so good so far, because I identify a lot of unspecific proteins (cytoskeletal proteins and so on). So I think i´ll have to improve my method. What things would you change?
1. This is my incubation buffer: 50mM Tris pH7, 150mM NaCl, 2mM EDTA, 0.2% Tritón X-100, 0.3% NP-40 (Igepal). I have read many protocols using MgCl2. What do you think about it?
2. My washing buffer: PBS, 1% Tritón x-100, 0.1% 2-ME. Should I use a similar buffer, such as the incubation buffer, with salts, to keep the interactions?
3. I incubate 4h lysate and GST-X. How much time do you do?
4. What´s your opinion about preclearing with sepharose, or blocking with BSA? Does it reduce significantly background?
5. What amount of GST-X and lysate do you employ?

I know it´s a lot of questions, but any kind of help will be highly appreciated.

Thanks in advance.


If your bait or protein needs metal ions such as Mg2+, Zn2+, Cu2+, Fe2+ to stabilize its structure, the metal ions should be added.
If your proteins contain disulfide bridges, 2-ME, or some other reducing reagents is needed in the procedures.

By the way, I have not performed GST pulldown yet, so my opinion is not solid.