finding accurate protein molecular weight - protocol? (Sep/07/2008 )
Hi,
I have synthesized a protein and ran it on an SDS-PAGE gel. On the gel, it does not run to the expected molecular weight. I was thinking of confirming its molecular weight by mass spectroscopy. Are there any protocols to do this? Has anyone had problems before where your protein does not go to the correct MW on a gel but other tools can confirm its weight?
Did you have the actual protein synthesized (e.g by solid phase peptide synthesis) or did you do it via a cell-free extract, or are you expressing it from a plasmid clone?
How far off is the molecular weight relative to your MW ladder? Which ladder did you use?
What are your SDS-PAGE conditions? What percentage gel did you use? What buffers? How big should your protein be?
I have synthesized a protein and ran it on an SDS-PAGE gel. On the gel, it does not run to the expected molecular weight. I was thinking of confirming its molecular weight by mass spectroscopy. Are there any protocols to do this? Has anyone had problems before where your protein does not go to the correct MW on a gel but other tools can confirm its weight?
single SDS-PAGE gels are not sufficient to determine precise molecular mass; you have to run several and calculate the average size of all runs; Ms is indeed more precise
A protocol for MS would depend on what MS instrumentation you have available. The simplest and most reliable would be ESI-MS (electrospray ionization). The sample prep is fairly simple- you'll just need around 20-50 uL or about 40-60 uM protein. Dilute the protein in ddH2O as ESI is sensitive to high salt concentrations. Some MS have a desalting column places before the injection port, so in these cases higher salt concentrations can be tolerated.
I hope this helps a bit... Good luck 