Login

Join for Free!
118822 members


SDS-PAGE analysis

Discussion of all aspects of cellular structure, physiology and communication.

Moderator: BioTeam

SDS-PAGE analysis

Postby 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,
madinacath
Garter
Garter
 
Posts: 3
Joined: Thu Jan 05, 2012 10:05 pm

Re: SDS-PAGE analysis

Postby 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.
User avatar
jonmoulton
Viper
Viper
 
Posts: 433
Joined: Fri Feb 15, 2008 5:38 pm
Location: Philomath, Oregon, USA

Postby 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
Garter
Garter
 
Posts: 3
Joined: Thu Jan 05, 2012 10:05 pm


Postby 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?
madinacath
Garter
Garter
 
Posts: 3
Joined: Thu Jan 05, 2012 10:05 pm

Postby 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.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5689
Joined: Mon Sep 14, 2009 7:12 pm


Return to Cell Biology

Who is online

Users browsing this forum: No registered users and 1 guest