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My gels look significantly different in MES (2-(N-morpholino)ethanesulfonic acid) and MOPS (3-(N-morpholino)propanesulfonic acid). That is to be expected. What I don't understand is why the simple presence of the counter-ion significantly changes the velocity of certain proteins.

Is this due to the different pH (MES is usually run at pH 8.0, MOPS is usually run at pH 7.0) or is the charge distribution on the counter-ion responsible for everything?

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I'm pretty sure the net effect is caused by the charge distribution of the counter-ion. This will have the most immediate effect on the net electric field and therefore directly effect the electrophoretic velocity of the proteins. The pH of the buffer indirectly affects this because it will determine the net ion balance based on the pKa values of MES/MOPS, so you will get different results using the same buffers but at different pH values. –  leonardo Apr 6 '12 at 4:10
    
@leonardo. I feel that you're correct. Now that I'm looking at the chemical structure, I'm surprised that the single carbon difference creates that much difference in the pKa. However, if one is running a gel at the suggested pH for the buffers, the charge ratio should be the same. –  bobthejoe Apr 6 '12 at 4:14
    
these are non denaturing protein gels (No SDS)? –  shigeta May 14 '12 at 20:21

1 Answer 1

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+50

Well, to rationalize everyone's comments, I think @leonardo is right.

This is a denaturing SDA PAGE gel. The migration of the SDS Micelles which are negatively charged, depends upon the shielding of the solution around it. The difference in mobility is because the SDS micelles will experience a slightly different field at pH ~6.2 (MES) vs 7.2 (MOPS).

The thought that these have the same charge would be right at exactly the pH corresponding to the pKa. For these two ions the proportions will not be the same when running 0.8 pH units from their respective pKas. the ion concentration goes as +/- log ([BH]/[B-]) and the charge environment of the buffer should not be the same. I think that the higher pH for MOPS running will tend to create more negative charge in the solution from MOPS- but also OH- in solution, slowing the mobility of the micelles and favoring the resolution of the larger proteins with MOPS.

This is all given even if the buffering ion concentration is the same.

I'm sure the gory details are buried in the musty tomes of some physical biochemistry journals deep in the library, but this is how it sounds to me.

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Congrats, you get my reputation. @Leonardo gets props for showing us the way. –  bobthejoe May 15 '12 at 0:32
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I'm glad you could do the leg work to construct an answer for us. :) –  leonardo May 15 '12 at 2:14

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