I am performing immunoprecipitation (pulldown) of proteins using Ni-NTA agarose resins. My test control showed that the target/prey protein binds to the Ni-NTA agarose resin in the absence of the bait protein possibly due to the sticky nature of my target/prey protein. I have tried different buffers with different pH and some detergents but none seems to give a better result.
What is/are the best buffer(s) used to pulldown sticky proteins? Thanks!
If it is an ionic interaction, increasing the salt concentration may help. If it is a hydrophobic interaction reducing the salt concentration and adding a nonionic detergent may help.
Hi there,
I've always included 1% Nonidet P-40 in CoIP buffer. My basic recipe is:
50mM Tris–HCl, pH 7.5, 150mM NaCl, 1%Nonidet P-40 supplemented with Complete EDTA-free anti-protease cocktail. Indeed NaCl concentration may be adjusted.
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