I need to separate different forms of a protein around 36 kDa: non-glycosylated, mono-glycosylated, di-glycosylated, tri-glycosilated and tetra-glycosylated.
My goal is to detect these proteins by western blot, using an anti-GlcNAc.
I am thinking to use a polyacrylamide gel between 12-15 % and a system bigger than mini-protean. Any advice or suggestions?
Dear Sandra,
Since one glycosylated site gives you up to 2kDa difference you may expect that if you make a large (size) gradient (I would start from 10 and finish with 15%) gel and run it as Magdalena proposed you will see a nice difference after staining. I think it is reasonable to use a comb, making wide pits, and when you will develop the blot try to adjust the exposure time to yield the thinnest bands possible. I think it should work.
Good luck,
Alexey
I agree with Alexei using a comb with wider pits... 10 - 15 % gradient sounds reasonable but you can also just use 12 % gel and run it until the end, half of the gel seperates between 10 - 20 kDA for example
Do you mean four different sites of glycolysation? each of ones with one Glc?
Hi, I agree with all of them, but if you continue having problems, you can try to perform a 2DE gel and testing different isoelectrofocusing strips, or even you can use the liquid isoelectrofocusing (IPGPHOR) for trying to seperate perfectly the isoforms, but this can be hard.
Good luck
Hey,
You can try to increase the voltage difference between stacking & resolving gel. As my experience tells if the samples are stacked well, their resolution becomes better.I generally prefer 60V for stacking and 75 V for resolving gel. Moreover, increasing the %age of Acrylamide and Bisacrylamide will majorly lead to the shift in position of your protein on the gel. I am skeptic about the improvement in resolution.
Good Luck.
I agree with Felix, if you want to separate different isofoprms, the best choice is the 2DE. You can focus your protein by changing the range of IPG strips, for the second dimension you can try with 4-12% A/BA.
You can detect directly with better mass accuracy on a MALDI-TOF using peptide mass fingerprinting (PMF) after deglycosylating with PNGase F. In a case where this is not available, try GCE on-a-chip or carefully follow the great suggestions above.
there is no (or a margin ) chance to resolve about 200 Da differences on gel. go for IEF, might be work (you can blotted it too, i think, some care). other way, this is not a question of any kind of page. moreover, glycolysation results smearing on gel usually... good luck.
I would expect a 15 % acrylamide Lämli-Gel to do the job. Probably you can use a protein ladder mix for small proteins that distinguishes well between protein sizes in the range you expect to detect your proteins. I would then also run the gel until the ladder bands you don´t need to detect (lets say until 26 kDa) run out of the gel, because you increase the separation distance. I agree with others who suggested to separate at a low voltage not higher than 80 volt. With such a technique I also manage to distinguish between mutant and wildtype SOD1 which share the same protein size but reveal slightly different running behaviour in gels for an unknown reason. Some people believe running the gel on ice or in a cold room improves resolution. Might be worth a try! Good luck!
Try to reduce the size of the gel system, although sacrificing separation distance of the resulting banding pattern. The smaller the gel, the greater the concentration of each protein band, but the closer the bands will cluster together. If you run several identical smaller get systems, and pool the desired protein band and then rerun a larger gel with the pooled band, you will not only have a very stark resulting band, but an almost pure one.
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