We precipitated the proteins using methanol-chloroform protocol and redissolved the obtained protein pellet in Tris/PBS buffers. But the proteins are not completely dissolved leaving behind a mass of undissolved proteins in colloidal form. Even-though we didn't keep the pellet drying for long time after the precipitation, still the proteins doesn't solubilise. We vortexed the samples after adding little formic acid and found that most of the proteins got dissolved.Will this be an issue if we are performing 2D gel electrophoresis and suggest the methods that we can use to solubilise those proteins.
Thank you very much
After the precipitation, the proteins were denatured. In addition, in your pellet, you might have hydrophobic protein. And even with hydrophilic proteins, the carbon skeleton of the protein is hydrophobic. After denaturation, if you do not add a detergent such as NP40, CHAPS or SDS, the proteins remain insoluble.
The addition of formic acid is not a good idea for 2D electrophoresis, due to salt and pH modification.
The easiest for 2DE, after your precipitation, is to recover the pellet in a 2DE lysis buffer with Urea, thiourea, CHAPS and DTT.
I'd assume they are simply denatured. That's the disadvantage of precipitation by organic solvents.
After the precipitation, the proteins were denatured. In addition, in your pellet, you might have hydrophobic protein. And even with hydrophilic proteins, the carbon skeleton of the protein is hydrophobic. After denaturation, if you do not add a detergent such as NP40, CHAPS or SDS, the proteins remain insoluble.
The addition of formic acid is not a good idea for 2D electrophoresis, due to salt and pH modification.
The easiest for 2DE, after your precipitation, is to recover the pellet in a 2DE lysis buffer with Urea, thiourea, CHAPS and DTT.
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