Protein-protein binding buffer

[Andreas "Mega" Stuermer]

Hi guys,

I want a peptide to bind to a protein.

After I extracted the protein from the organism (extraction buffer 0.1M Tris-HCl, 0.1M NaCl, pH 8) I will put them into a MWCO centrifuge thing to remove small molecules  with 3 kDa cut-off (everything smaller than 30 amino acids flows through the filter and is discarded).

What buffer should I put the protein and the peptide in? I know they react under physiological conditions in the cytosol. Maybe I should not use extraction buffer at all and just use the cell pulp plus protease inhibitors?

Looking forward to some input

[Andreas "Mega" Stuermer]

Will deionized water work, maybe? If I do 1% extraction buffer and 99% dH2O, the pH might still be stable (measure it) and there's not much NaCl to prevent binding of the partners

[Bryan Jones]

While it's always dependant on the specific protein and peptide, a good place to start would be with something physiological-ish, like the extraction buffer or PBS. Depending upon how tightly your protein and peptide bind and how specific you need the interaction to be, you might want to add a bit of detergent to prevent nonspecific interactions. With antibody binding the standard is PBS with 0.1% tween20.

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[Andreas Stuermer]

So you're saying PBS is probably good? I thought the salts may prevent binding

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[Bryan Jones]

The salt in PBS won't prevent binding if it's a strong interaction. If you have a very weak or transient binding it might be something to consider (maybe use 10% PBS), but if the protein binds the peptide in vivo, then the similar salt concentration in PBS shouldn't interrupt that binding. But, again, it depends on your specific protein/peptide.

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[Andreas "Mega" Stuermer]

Perfect, highly appreciated. Yeah i guess that makes sense, will try PBS