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Promega IVT Problems


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#1 Chipper

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Posted 12 October 2010 - 07:08 AM

Hello
I am using Promega's TNT in vitro transcription/translation system (rabbit reticulocyte) to express a protein (s35 labeled) and look at its interaction via pulldown with another (gst)protein. The IVT itself always works but my constructs express protein about 10x less than the luc control provided by Promega. Does anyone have any advice or suggestions for getting a stronger IVT product?

Currently I'm using qiagen columns to purify the pasmid.

Thanks

#2 David C H

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Posted 13 October 2010 - 07:27 AM

You could try switching to a wheat germ kit with a feeder system (from Roche, I believe, but they may have sold this kit to 5 Prime). The reaction time is longer, but it is the only eukaryote kit that can produce the same yields as the prokaryote kits. You may be able to mix the rabbit kit with an S30 e.coli kit similar to what was done here:

http://www.proteomes...8/1/32/abstract

That was done using Promega's wheat germ kit -- but as I recall, it doesn't work with the rabbit kit when components are mixed in a 50/50 ratio (so a different ratio might work for the rabbit kit).


Hello
I am using Promega's TNT in vitro transcription/translation system (rabbit reticulocyte) to express a protein (s35 labeled) and look at its interaction via pulldown with another (gst)protein. The IVT itself always works but my constructs express protein about 10x less than the luc control provided by Promega. Does anyone have any advice or suggestions for getting a stronger IVT product?

Currently I'm using qiagen columns to purify the pasmid.

Thanks






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