I want to do a pulldown using 2 purified proteins. One is Gst-tag, the second is His-tag. I want to use the immobilise the Gst-tag protein on glutathione sepharose 4B beads, and try pulldown the His-protein (prey).
What would be a suitable concentration to use for the prey protein? I will use 5ug of the Gst protein. I tried using 5ug prey protein but I get non-specific bindings in all my samples including negative controls (Gst only + beads; beads only).
Hi there,
If you get non specific binding of your prey protein, it means the stringency is not high enough. Depending on your current wash conditions, you can increase Np-40 concentration and/or ionic strength (NaCl). Hope it helps.
Hi Howard,
If you got non specific binding, you won't be able to get any valuable information from the experiment. The first thing I would try is to make the pull-down in reverse way (with His-Tagged protein immobilized and 20 (to 40 mM) Imidazole).
Second, if you want to improve the same experiment, you need to get the correct buffer (notably the Glutathion concentration, and also usually salt concentration) at wich the unspecific binding is minimal, so that the signal you see with your immobilized protein means something in terms of binding of the second protein.
Hope this helps,
YV
Hi everyone, thank you for your replies. Making a more stringent wash buffer helped rid the non-specific bindings. The wash buffer was:
20mM Tris, 200mM NaCl, 5mM DTT, 1% Triton X-100
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