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Denaturing protein


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#1 scientist

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Posted 11 January 2005 - 10:46 AM

I am working with a protein that has a theortical size of 17kDa. It has 3 isoforms that are theoretically capable of binding together. There are no cyseine residues in the protein but it has a leucine zipper. We presume the isoforms bind together through the zipper.

We are trying to do Westerns and I have treated the sample with SDS/beta-mercaptoethanol. The protein size on the gel is approximately 46kDa but we can never see the 17kDa fraction.

Potentially I have 2 problems: 1) an antibody that does not work such that it is not specific; 2) an antibody that is correct but I cannot get the protein to fall apart.

Would you have any suggestions for alternatives on denaturing proteins?

Edited by scientist, 11 January 2005 - 10:47 AM.

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#2 eleceyes

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Posted 11 January 2005 - 02:32 PM

Which transfer membrane did you use for your western blot?
Some membranes are specially designed for small proteins (<20 kDa).
I also find the blotting condition is very crucial too!

Does your protein present in abundance?
If not, you might want to try a more sensitive detection system (e.g. ECL-Plus instead of ECL).

Hope this helps.

#3 scientist

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Posted 11 January 2005 - 07:21 PM

Thank you for the reply eleceyes. I use a PVDF membrane for blotting. I did wonder if that was a problem but I can see the ladder at 14.3 kDa. So I am not sure. Theoretically, if I can see 14.3kDa then I should be able to see a 17kDa protein as well.

The protein is not that abundant.

I am now wondering about glycosylation or other sort of mechanism that will make the protein larger than predicted and not allow it to fall apart.
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