2DE of hydrophobic membrane protein (problem) - (Jan/08/2007 )

Hi!
I have problem with one membrane protein from Escherichia coli - highly hydrophobic one. It has theoretical pI value 9,3; MW about 35 kDa and the GRAVY Score is +1,09. Does anyone know if it's possible to obtain the spot of that protein on 2DE Gel? If so, what are the ways of preparing such a hydrophobic sample and what are the detailed conditions of 2DE? The "usual" protocols don't work at all... :-(

you can extend the pH range of the ief gel by the addition of pH 8-10.5 ampholytes.

hi there,

there are quite a few protocols available that deal with buffer conditions for IEF of hydrophobic proteins, though you might need to do perform quite a few of them to optimize for your protein of interest. i'm guessing you're looking to run the 2D gel for some sort of protein identification project. if that's the case, you might also want to consider another sort of separation besides IEF. some sort of reverse phase separation on C4 resin or hydrophobic interaction chromatography followed by conventional SDS-PAGE might be of benefit for you. often, researchers lose a considerable amount of hydrophobic protein in their IEF separations that might otherwise have been found had they chosen a separation technique better-suited to the biochemical properties of membrane proteins.

that being said, if you absolutely must use IEF to isolate/identify this protein, you might consider enriching for your basic proteins in a preparative IEF cell like the Rotofor or Invitrogen's Zoom fractionator, and then load the basic fractions on a narrow range IPG strip (i think some companies make them from 9 - 12, or something along those lines). you're probably going to lose a bit of your protein in the IPG strip due to both it's pI and its hydrophobicity. while it's possible your protein will precipitate in any of the above IEF separations, you stand to have a better chance of finding your protein this way. considering that you're purifying this from E. coli, you shouldn't have too much of a problem getting enough protein to try a multitude of approaches.

lastly, i'm not sure what your protocol is for sample cleanup, but be sure to remove the lipids from the membrane. there are a number of protocols out there for this, quite a few of them involving the use of methanol/chloroform.


good luck