GST pulldown assay problem - (Jul/23/2007 )
I have a question on one of my result about GST pulldown assay.
the question is when i used GST fusion proteins to pulldown the HA-tagged prey proteins translated in vitro, i got a band that has higher molecular weight than the HA-prey, which is detected by anti-HA in western blot. So, is it possible that the band detected is actually a complex of GST-bait and HA-prey?
Thanks for any replies!
I tried the same experiment and also had background bands well above the molecular weight of my in vitro translated product. My background bands were around 50 and 25Kda. If you ran a SDS gel the band you are seeing is not a complex as SDS is a denaturing gel and will break interactions. You may want to run a lane of the negative control TNT (no DNA was added to the in vitro reaction) to see if this is background from the in vitro system. Do you see an HA band at the correct size as well as this band, or just this higher molecular weight band? If it's just the one, you might want to make sure you have a proper stop codon.
Thanks for your suggestions!