Gel Electrophoresis at a specific pH

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Aloha
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Gel Electrophoresis at a specific pH

Post by Aloha » Mon Apr 24, 2006 9:49 am

Hi all

Need to answer following question:
A sample of solution X is examined by gel polyacrylamide electrophoresis at pH 7.0 (with no SDS presents). After staining 6 bands showed up. The same procedure was repeated but at pH 10.0. This time only 4 bands showed up. Why is there difference in pattern?

All readings I've done always speaks about gel electrophoresis with SDS presents. However I suggest since there is a pH, maybe at pH 7.0 there are less proteins with the same isoelectric point whereas at pH 10.0 there are more proteins with the same isoelectric points. And since a protein with its isoeletric point corresponding at pH 10.0 in a solution with pH 10.0 would be inmobile. But then there is no pH gradient so.....hmmm I'm getting confused now.

Are my thoughts correct or is there anything else why the pattern of bands differ at different pH's in gel electrophoresis? Hope someone can help me.

Cheers
Aloha

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LilKim
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Post by LilKim » Tue Apr 25, 2006 12:16 am

this question has to do with protein structure... SDS is a detergent that gives a protein a net overall negative charge ... therfore, molecules cannot migrate differently due to their inherent charge (pi)... therefore you don't have to worry about thing migrating differently due to isoelectric points.

So, if you're going to run a protein through a gel (using electricity).. the pH of the buffering solution and the proteins inherent pi will affect the mobility ( I would assume).

I'm not exactly sure how this works (someone explained this to me the otherday ... obviouslyy I don't understand it as well as I should .. but hopefully this mayy help you a bit.

buena suerte!
- KIM

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