binding buffer in GST-pulldown - (Apr/10/2007 )
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 which is sonicated to gain the protein;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)
Hi, sorry I did something wrong with the previous post.
Most protocols I´ve read use physiological salt conditions, but also add Mg+2 sources and non-ionic detergents. I incubate the binding in a smaller volume than following washes. Temperature of incubation might affect as well the binding efficiency of your proteins. One last thing: are you sure that your purified GST construct is native after purification?
Here is a recipe that worked fine for me:
150mM KCl, 20mM HEPES, 0.1%NP-40, 5mM MgCl2, 10% Glycerol, 1mM DTT, 1mM PMSF, 0.5mM EDTA
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 which is sonicated to gain the protein;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)