GST pull down assay - (Mar/19/2009 )
I am trying to do a GST pull down assay. I have the GST-fusion protein successfully produced in EColi cells. I also have successfully expressed my protein of interest in HEK293 cells.
I have several GST tagged proteins. I want to see which if any of them bind to my protein of interest. I did a library screening using yeast two hybrid throwing my protein of interest. The GST pull down is to confirm that the binding is indeed possible.
I need to now design several setups and then attempt the pull down. What controls should I have to make sure my results are indisputable?
Has anybody else done something similiar to what I am doing?
You need a beads-only control, a GST-only control, a non-specific antibody control and preferably a positive control. I have a feeling I am missing a control there too, check out "current protocols in molecular biology" for a good guide.
bob1 on Mar 19 2009, 04:07 PM said:
Thanks for your help. what do you mean by non-specific antibody control?
An antibody that is not involved in the system you are looking at, to see if just any old antibody could have pulled down the proteins you get from your IPs.
bob1 on Mar 22 2009, 03:51 PM said:
I didnt do an IP. I did a yeast 2 hybrid library screening. What would be a good Ab to start with?