Thread: GST-pulldown

Hi,guys

I'm now doing GST-pulldown to detect the interaction between two known protein.But there's no binding detected.

One of my proteins is fused with GST,and expressed in E.coli;the other is translated labeled with S35-Met in vitro in TnT system.I'm sure there's nothing wrong with protein synthesis.But still there's no binding.Could anyone tell me what's wrong?How to ensure there's no binding?

Does it have sth. to do with the binding buffer?what's the normal binding buffer in GST-pull down?(I have used PBS or 150mM NaCl as binding buffer)

For a control you should put your construct behind a LacZ promoter so you can induce its expression. Then test it and see if your purification is working.

Or something along those lines.

http://www.protocol-online.org/cgi-b...GST&submit2=Go <-- that website may have your answer.

Thanks ,Sig

However,I'm sure there's nothing wrong with the GST-fusion protein purification,at least on the expression level.I've collected the liquid of each step and done Coomassie Blue Stain,the MW of the bands is right.Of course,there could be conformation change problem,but I don't know how to detect.

I preserve the fusion protein in PBS, and freeze in -80°C