Lost Protein?

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Lost Protein?

Post by BrianScott » Tue Jun 30, 2009 4:05 pm

Looking for advice on my current situation.

I have confirmed the recombinant protein I'm working to collect is expressed in T7 ecoli/pET24a. And after addition of lysozyme and then sonication the supernatant also showed the expected protein. INFO: the protein's iso electric point is predicted to be 9.8... the solution for the column is 20mM TRIS(pH 8.5)... and I salt out using KCl across the concentration gradient of 0.1M - 1.0M... and the column is a anion exchanger, sepharose (Q). Now the problem is I'm not seeing my protein after elution, roughly about 20 times the volume of the column has been passed through (again, 0.1M to 1.0M). The protein was supposed to come off with out having to salt... as predicted my the iso electric point and pH of the solution... so I'm not sure what is going on... except that I can't retrieve my protein.

Any advice you can offer on solution concentrations, pH... anything.

Side note:
When I add SDS loading buffer to the eluted samples a lot of white precipitate falls out... I've just been heating the sample for 10 min at 100degrees C and then spinning down the precipitate after the sample cooled down. Could this be a reason I'm not seeing my protein on the gels?


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