Protein purification

hdani21 at hotmail.com hdani21 at hotmail.com
Tue Mar 2 10:32:15 EST 2004


Hi, I am working with Tryptophan hydroxylase protein. The gene is expressed in pET21D vector by fusing the His tag to it. I got the soluble protein. But whenever I run an analytical get to see the soluble fraction, it shows me smaller size than the induced protein. Why soluble fraction does not show the same size as induced one? 

I am using Ni-NTA affinity chromatography to purify the soluble protein. It shows the peak during the wash but it does not elute the protein. I don't get any peak when I do the elution. I am using Tris buffer for the purification. And also I read the absorbance at 280nm. Is it right nanometer to read the elution peak? And how one decide at which nanometer one needs to read the elution?

Thank you.

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