I am trying to solubilise bacteriorhodopsin that I have purchased from Sigma (product code B0184) and was hoping that someone out there may have experience working with this protein. Ideally, I'd like to solubilise it such that I can proceed directly to tryptic digestion. I have tried using 60% methanol:40% ammonium bicarbonate (final concentration 50 mM) in accordance with papers on bacteriorhodopsin solubilisation (Blonder et al., Proteomics, 2004, 4, 31-45) and tryptic digestion in organic solvents (Russell et al., Anal. Chem., 2001, 2682-2685). I was hoping to follow this approach as a) a cost-effective protocol not requiring reagents like RapiGest, and b) a clean protocol such that the only post-digestion manipulation is the removal of organic solvent by vacuum centrifugation. However, using this approach, there was still solid material after addition of solvent, which was yellow-orange in colour. Is this protein-related? The amount of solid stayed the same after overnight digestion with trypsin (1:50 enzyme:substrate), so I was thinking perhaps it isnâ€™t? However, I have no experience working with membrane proteins, so Iâ€™m not sure what to expect. Can anyone offer any advice in terms of preparing bacteriorhodopsin for MS analysis? Would sonication be advisable to aid solubilisation?
Many thanks in advance,
Share protocols and ask for sample preparation advice.
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