I have been doing pulldown with bacterial purified GST tagged protein as bait and HEK293T expressed Ha tagged TRIM family proteins as prey. I prepare whole cell lysate in a buffer containing mild detergent (NP40), almost 48 hours post-transfection. Since, GST tagged proteins are preys, I have only GST as negative control for interaction.
So, the problem is that I get a huge band in GST control as well which is of similar molecular weight as preys. I tried pre-clearing the whole cell lysate but no luck so far.
Also, I would like to inform that I do not elute with glutathione, I just simply boil beads after last wash in SDS loading buffer, centrifuge and load on SDS-PAGE. Any sort of suggestion would be help full.
Could you clarify which one is prey? you mentioned both GST tagged proteins and TRIMs.
GST tagged MD-D2 protein is bait and Ha tagged TRIM 25 (and several other TRIMs) is prey. I use 0.05 % NP40 and 50 mM NaCl in interaction buffer (which I use as wash buffer as well). I just worried if I make buffer a bit more stringent, I might end up loosing target interaction as well.
GS transferase has a very strong affinity for GST beads (up to 1% NP40 and 1 M NaCl, perhaps higher), don't worry about it. What I'd worry more about is your bait to prey affinity that may be compromised at high salt. Are you detecting with HA? My understanding is that you are. You could be picking up your GST (26 kDa)/or GST -fusion (size?) band simply because its sheer abundance makes it detectable by non-specific interactions with the detection antibodies . If that is the case (to check, just stain your blot with your favorite reversible protein dye) you should consider eluting with high salt only : assuming ionic interation, this should disrupt your prey/bait interaction but not your bait/bead interaction. Hopes this helps
good luck!
Thank you Sebestien for the help. I will take suggestions into consideration.
Agree with Soubeirand suggestion. Try it and tell us if problem is solved. Jorge.
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