I am interested in isolating two isoforms of molluscan hemocyanin which are 3-4 MDa. There is very little difference in their molecular weights (only a handful of AA changes). But they are known to have isoelectrical points varying around 0.3 or so.
What would be the best method to assess this? Can 2DE handle such large MWs especially with such small differences in MW?
Hi Matthew, have you tried this Biorad system called Rotofor? The great advantage of this apparatus is that allows separations of proteins (in solution) with small differences in PI . The trick is find the correct ampholyte range to get the higher resolution. Of course you can use it for preparative native preparations. Best of luck.
Hi Matthew, I think the question comes down to the scale of the proteins you want.
Fernando is correct that you could use preparative IF (Rotofor) to isolate mg quantities of the proteins. I have used Rotofor before and it is a trial/error procedure,like Fernando describes.
If you need small quantities (~1-100ug) you could try native 7.5% PAGE. Alternatively you could use agarose electrophoresis.
Hi Matthew, here are some references for a quite old but robust method for preparative isoelectric focusing from Bertold J. Radola that might help you:
1. "Analytical and preparative isoelectric focusing in gel-stabilized layers."
Radola BJ. Ann N Y Acad Sci. 1973 Jun 15;209:127-43.
2. "Isoelectric focusing in layers of granulated gels. I. Thin-layer isoelectric focusing of proteins."
Radola BJ. Biochim Biophys Acta. 1973 Feb 21;295(2):412-28.
3. "Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing."
Radola BJ. Biochim Biophys Acta. 1975 Mar 28;386(1):181-95.
Best, Murat
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