problem in GST pulldown

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jasfire
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problem in GST pulldown

Post by jasfire » Tue Apr 10, 2007 3:50 pm

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)

blcr11
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Post by blcr11 » Tue Apr 10, 2007 5:07 pm

I would have thought PBS would have been fine. The GST-Bind kit from Novagen has as its loading buffer a phosphate buffer at pH 7.3 that contains 137 mM NaCl and 2.7 mM KCl. Don't know if the KCl is critical or not.

Did you have a positive control that can bind labled protein--and it worked? What I'm getting at is whether you know for sure that the S35 incorporation actually worked because, if it didn't, you won't see anything either.

jasfire
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Post by jasfire » Tue Apr 17, 2007 3:56 pm

the S is out-of-date(about 2 month),but the free isotope could be detected.Does that mean the isotope still can work?

blcr11
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Post by blcr11 » Tue Apr 17, 2007 5:43 pm

I couldn't swear that it won't work, but I'd guess that you're at least out by 1-2 half-lives on the isotope, depending on the "fresh" date. The half-life of S35 is 87 days. If you're out 2 half-lives the radioactivity is about a fourth of what it was on its "fresh" date. If it were me, I'd prefer to use fresher isotope, but it may work anyway. You might try and over-expose your blot to see if you can see anything at all.

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