SDS-PAGE analysis

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madinacath
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SDS-PAGE analysis

Post by madinacath » Thu Jan 05, 2012 10:16 pm

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,

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jonmoulton
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Re: SDS-PAGE analysis

Post by jonmoulton » Thu Jan 05, 2012 11:11 pm

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
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Post by madinacath » Thu Jan 05, 2012 11:27 pm

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
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Post by madinacath » Fri Jan 06, 2012 12:27 am

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?

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JackBean
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Post by JackBean » Fri Jan 06, 2012 4:04 pm

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)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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