Protein purification

dan su dan_su at hotmail.com
Tue Mar 2 12:06:18 EST 2004

Problem #1, not a problem, this happens frequently
Problem #2, what is in your wash buffer? Did you sequence the construct to 
make sure the construct right, are you sure there aren't any frame shift 
that may cause the loss of C-term His? And read QIAGEN's "The 
QIAexpressionist", you don't need to moniter the wash and elution, just run 
SDS-PAGE to visulize.

>From: hdani21 at hotmail.com
>Reply-To: hdani21 at hotmail.com
>To: proteins at hgmp.mrc.ac.uk
>Subject: Protein purification
>Date: 2 Mar 2004 15:32:15 -0000
>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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