why did the membrane protein disapear again? - (Mar/30/2007 )

Hi everybody,

Since fast 3 months I have been trying to purify the membrane protein, it is about 30kDa, tagged with His6 and have six transmembrane helice, which is expressed in E.coli . After the overproduction I have made a preparative gel which showed that the protein I want is indeed there. Then I have tried to purify the protein by His-tag chromatography, electroelution(after this step the protein is still there) and RP-HPLC(followed by vaccum trocken), after the last step , I found out that my protein disapeared! I precipatate the solution,no pellet produced. I have thought that the protein was perhaps stick on the tube wall , so I take the solution out of the tube and wasch the tube with SDS sample buffer, but at last it turned out that nothing is on the tube wall. Where is my protein? I will be very appreciated if you can give me some suggestion.
Thanks
Linda

Was it still present after the RP-HPLC? Did you consider trying hydrophobic interaction chromatography?

[quote name='swanny' date='Apr 2 2007, 03:45 AM' post='92894']
Was it still present after the RP-HPLC? Did you consider trying hydrophobic interaction chromatography?
[/quote)

Thanks for your reply. Because the presence of isopropanol(resulted from RP-HPLC) in the sample it is very difficult to load the sample into the gel, so I have to dry the sample first. The RP-HPLC is already the hydrophobic interaction chromatography.