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common reasons proteins show up at different sizes on blot


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#1 Curtis

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Posted 03 February 2011 - 08:25 AM

The viral protein I am working on is supposed to be seen at 42 kDa according to its NCBI sequence, but in reality it shows up a little higher around 49-50 kDa on western blot. what are the common reasons? phosphorylation?

Edited by Curtis, 03 February 2011 - 08:26 AM.


#2 knuf

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Posted 03 February 2011 - 10:41 AM

Yes, phosphorylation can cause your apparent molecular weight to be off--as well as other post-translational modifications. One thing that is often forgotten in SDS page is that it separates based on both size AND CHARGE, and while it is generally assumed that SDS coats proteins equally, thus giving each the same negative charge, this isn't always the case (see work with Tau protein, which is known to run at a different apparent molecular weight than its amino acid composition would suggest). Also, if the protein is not completely reduced, remaining dilsulfide bonds can cause the protein to run aberrantly by altering the molecular size of the protein (which incorporates both weight and length). Finally the lipophilicity of the protein can affect the apparent weight, which i guess is due to the charge similar to SDS coating--see work with LC3 in the autophagy field--when the protein is conjugated to a lipid molecule, although the actual molecular weight is higher, it runs smaller due to the added lipophilicity.

Edited by kfunk106, 03 February 2011 - 10:44 AM.





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