SDS-PAGE analysis

[madinacath]

I need to find the number of n-linked oligosaccharides in different secreted proteins. I've completed an SDS-PAGE experiment and have a set of results. I've drawn a calibration curve of the molecular marker and therefore have an estimate of the MW of the different proteins before and after they have been digested with PNGase F. ..any ideas on how I find the number of N-linked oligosaccharides from this?

Thanks,

[jonmoulton]

Estimate the average mass of an n-linked oligosaccharides. Let that mass = NLOmass (g/mole)

Subtract the mass of the protein after PNGase F treatment from the mass of the untreated protein. Let that mass = DiffMass (g/mole).

DiffMass/NLOmass = estimate of the number of oligosaccharides on the protein before digestion (dimensionless). Round to the nearest integer.

[madinacath]

Thank you, that's extremely helpful!

One question, which may be seem little stupid.. how do I estimate the NLOmass? I'm confused as to how I can use this to calculate the NLOmass, as there are so many different n-linked oligosaccharides?

Thank you in advance,

[madinacath]

ok, I don't think that made a great deal of sense..

I meant, how to I estimate the average mass of an n-linked oligosaccharide?

[JackBean]

I think the number of bound monosyccharides is quite narrow, isn't it? Consider they usually contain hexoses, which all weight the same (just don't forget there is a water substracted)