4 replies to this topic

[MaggieRoara]

Hi everybody.

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?


Thanks

[bob1]

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.

[MaggieRoara]

bob1, on Mar 19 2009, 04:07 PM, said:

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.

Thanks for your help. what do you mean by non-specific antibody control?

[bob1]

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.

[MaggieRoara]

bob1, on Mar 22 2009, 03:51 PM, said:

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.

I didnt do an IP. I did a yeast 2 hybrid library screening. What would be a good Ab to start with?