Improved deglycosylationI deglycosylate in Pierce Reacti-vials (0.3ml vol, glass w/ teflon covers).1- Add sample (5 to 20 micoliters) to open vials; leave overnight to evaporateNotes:
2- On ice, add 5 microliters of m-cresol, 20 microliters of trifluoromethanesulfonic acid, cap vials, mix
3- Incubate 10 min to 3 hrs at 0C (or RT)
4- Stop reaction with 250 microliters of cold acetone:N-ethylmorpholine (4:1 by volume)
5- Cap vials and mix; transfer solution to microfuge tubes. Add another 250 microliters of acetone, 2 microliters of 1% Ficoll (carrier), mix
6- Incubate 30 min at -20C, pellet protein 2 min at 14K. Wash pellet with 700 microliters of acetone; centrifuge again.
7- Dry pellet; take up in 20 micoliters of cracking buffer.
8- Proceed as usual with SDS gel electrophoresis
N-ethylmorpholinium trifluoromethanesulfonate is a solid (inconvenient to dissolve). Adding the N-ethylmorpholine in cold solvent prevents solidification and more effectively cools the neutralization (highly exothermic).
Phenol (water soluble protein solvent) is reported to be an effective scavanger. M-cresol is more convenient because it's a liquid.
Ficoll is a convenient carrier for precipitations.
Phycodnavirus glycoproteins show no microheterogeneity and are therefore better deglycosylation models.
Scavenger (cresol) helpful.
Higher temperatures speed up deglycosylation, but degrade products
Microfuge tubes are unsatisfactory for deglycosylation. TFMSA appears to extract material from tube walls.
Useful review: Edge, Biochem J 376, 339-350 (2003) - Deglycosylation of glycoproteins with trifluoromethanesulphonic acid: elucidation of molecular structure and function.