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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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phosphorylation/protein size?
Posted by: ianbirmingham (IP Hidden, New member, 7)
Date: July 5, 2006 09:39AM
Hi, I am doing invitro kinase reactions using purified recombinant proteins. After the reaction I resolve the proteins by SDS PAGE. My substrate is about 40KDa, however I see little if any phosphorylation of this major band, what I do see is a phosphorylated band running FASTER ie about 33KDa. The phosphorylated band is specific to lanes containing my substrate. Is it possible that phosphorylation could cause a protein to migrate faster than its unphosphorylated form?
Re: phosphorylation/protein size?
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: July 5, 2006 07:03PM
Phosphorylation will only cause slower migration because of extra phospho groups AFAIK. What substrate protein are you using? If it is pure enough it should give one band with little higher position than the unphosphorylated substrate.
Re: phosphorylation/protein size?
Posted by: ianbirmingham (IP Hidden, New member, 7)
Date: July 6, 2006 03:24AM
I would agree I would expect proteins to run slower, but SDS separation relies on negative charge thus could it be possible that the extra -ve charge could cause faster migration? In answer to your question, I have a reasonable pure coomassie band, but running ahead of it by about 5Kda, when the autorad is aligned, is my phospho band.
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