Separation of similar weight protiens - (Jan/30/2008 )
Can someone tell me if I am on the right track here, I'm only learning!
Age separates proteins by there ability to move through the media so charge on aa's size and shape all play a part
sds-page should take the protien down to primary structure and give it a nice even charge depending on it's aa length (therefore mw on average-ish depending on base composition).
>So if the protiens are of similar molec weight I may not see the difference with sds-page
>And if my globular protein is rich in cyt residues it may not go down to primary structure, so I get lots of bands at the bottom of my gel
Thanks in advance
Age separates proteins by there ability to move through the media so charge on aa's size and shape all play a part
sds-page should take the protein down to primary structure and give it a nice even charge depending on it's aa length (therefore mw on average-ish depending on base composition).
>So if the protiens are of similar molec weight I may not see the difference with sds-page
correct
it will still be large and retarded by the matrix so it should not just go to the bottom of the gel. how cyt rich would it have to be to have that much effect?
are you using agarose (age) or polyacrylamide (page)? polyacrylamide is the matrix of choice for protein electrophoresis.
it will still be large and retarded by the matrix so it should not just go to the bottom of the gel. how cyt rich would it have to be to have that much effect?
>seems like a rhetorical question? I haven't a clue!
are you using agarose (age) or polyacrylamide (page)? polyacrylamide is the matrix of choice for protein electrophoresis.
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>Both, AGE shows differentiation between the proteins I am comparing, sds-page does not I'm trying to understand why so I can get it to work.
sds-page won't show the charge differences that you are seeing in age because, as you pointed out in your opening post, sds coats proteins with an even charge (density). this nullifies any charge differences that you wish to exploit.
you can, however, use native page. with native page you can see some charge differences. you can find some formulations in bioforum posts, just search.
if you wish to enhance charge differences then you can use urea page. we used to use this method to look at phosphorylation states of proteins. a one phosphate difference caused significant change in migration.
Thank you very much for your help! 
Know what to research now, which always helps.
Have a beer on me!
Thanks in advance
If you want to get to primary structure and have lots of Cys that you want reduced, remember to use DTT or B-ME in the loading buffer. That way you should avoid the problem of multiple bands (assuming you have just the one protein!!!)
Age separates proteins by there ability to move through the media so charge on aa's size and shape all play a part
sds-page should take the protien down to primary structure and give it a nice even charge depending on it's aa length (therefore mw on average-ish depending on base composition).
>So if the protiens are of similar molec weight I may not see the difference with sds-page
>And if my globular protein is rich in cyt residues it may not go down to primary structure, so I get lots of bands at the bottom of my gel
Thanks in advance
If you are testing two different proteins and you have separate Ab to detect them, then just divide the sample into two identical part and running on same gel, after that you can cut the membrane into two part and do western separately.
If you are testing two alleles (WT and mutant) with same Ab, you might want to tag one or both of them then do the similar way above.