How would you increase separation on PAGE? - (May/02/2006 )
my protein has 2 bands very close 88KDa and 93 KDa which were separated beautifully on SDS-PAGE....but i want to run my protein on urea-PAGE..and 2 bands came out as one fat band 
...forget about urea PAGE just thinking in terms of ANY PAGE what would you do to seperate those....decrease voltage....add DTT....decrease % of the gel (currently its 7.5%).???? please all suggestions are GREATLY appreciated.....cause im so so short of time..... 
...forget about urea PAGE just thinking in terms of ANY PAGE what would you do to seperate those....decrease voltage....add DTT....decrease % of the gel (currently its 7.5%).???? please all suggestions are GREATLY appreciated.....cause im so so short of time..... May try Gradient gel.
Don't decrease DTT, I think it won't do anything good.
hi
i agree with the previous posts and would recommend a 4-15% gradient gel.
...forget about urea PAGE just thinking in terms of ANY PAGE what would you do to seperate those....decrease voltage....add DTT....decrease % of the gel (currently its 7.5%).???? please all suggestions are GREATLY appreciated.....cause im so so short of time..... does the fat band appear near the top, middle or bottom of the gel?
if it is near the top then decreasing the acrylamide concentration would help.
if it is near the middle then increasing the concentration to ~10-12% should improve your resolution (this is what i expect you see).
if it is near the bottom then increasing the concentration to ~15% should work.
while a gradient gel improves overall resolution, if you are separating only the 2 bands identified in your question then a single concentration or shallow gradient (eg- 10-12%) gel is all you need and will resolve the two proteins better.
I agree with mdfenko, a gradient would be suitable to see all proteins from low to high molecular weight rather to separate to close bands.
do you run until front dye reaches the bottom? if your band is quite up, you could let it migrate more, until your band of interest migrates to the last third of the gel (I mean close to the bottom).
thank you so much for your replies!
i dont want to decrease DTT, i didnt add it, im thinking if I should add it...
i have attached the photo here....as you can see very poor resolution....sorry that marker (right) and my protein (left) have so much space in between...i wanted to load something in between but it didnt work out...
...so in the protein you might see two bands...lower one is not my interest....but the upper one (which should be two) is what i want to see clearly..in the marker two bands 97Kda and 66Kda are smashed together in one band (usually they are far away in SDS-PAGE..other bands are fine i guess..Missele,was also thinking about waiting a little bit longer, i turn off the gel when BPB reaches the end...how do i know for how long to wait?
N.B sorry it seems im not able to load the attachment anyway all the bands are in the most upper part of the gel....they are not even in the middle....
thank you for the suggestions again....
Don't decrease DTT, I think it won't do anything good.
i dont want to decrease DTT, i didnt add it, im thinking if I should add it...
..in the marker two bands 97Kda and 66Kda are smashed together in one band (usually they are far away in SDS-PAGE..other bands are fine i guess..
we use 5% (final concentration) 2-mercaptoethanol in our sample.
urea-page does not exhibit an exclusively size dependent separation, as with sds-page. there is a significant charge effect on the separation. we use the technique to separate a single protein with one or no phosphate attached and see a significant separation between the two. so i wouldn't expect size standards to run true (and, in fact, have seen them run 'wrong').
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urea-page does not exhibit an exclusively size dependent separation, as with sds-page. there is a significant charge effect on the separation. we use the technique to separate a single protein with one or no phosphate attached and see a significant separation between the two. so i wouldn't expect size standards to run true (and, in fact, have seen them run 'wrong').
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mdfenko, do you mean that I simply cant use UREA-PAGE to separate proiteins by weight? ...no matter what I do they just wont be separated clearly? 
...no matter what I do they just wont be separated clearly? not exactly. as with any porous material, urea-page exhibits a sieving effect, so molecule size is a consideration in the separation. the less restrictive the gel matrix is, the more influential the charge of the protein is on the resolution.
in an earlier post i pointed out that an increase in acrylamide concentration (which will decrease the pore size and make the matrix more restrictive) will allow you to make size a more important factor in the separation of your proteins. you should be able to improve the separation.
btw, when i said, in the previous post, that the standards didn't run 'true', i meant that the distance between bands did not properly reflect their respective sizes, not that they appeared in the wrong order (they were too disparate in size to do that). sorry for any confusion that statement may have caused.