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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Glycoprotein and Western Blot!
Posted by: teh (IP Hidden, New member, 1)
Date: May 11, 2006 07:22PM
Hi,
I'm expressing recombinant glycoprotein in Pichia expression system. Recently, a smear was appeared after Western blotting. Accoding to a paper, glycoprotein would appear as diffuse band on SDS-PAGE due to its glycosylated levels. How I prove that those are isoforms of glycoproteins, not unspecific proteins? I wonder to sent my samples for MS service or N-termination sequencing, any suggestion which labs are provided these service? I'm from Malaysia, so nearest is best! Thanks! Have a nice day! Teh
Re: Glycoprotein and Western Blot!
Posted by: arrohw (IP Hidden, New member, 1)
Date: May 30, 2006 01:24AM
Try a digest of your proteins with EndoH before starting SDS-electrophoresis. This should cleave the sugar-chains. The result would be a reduced molecular weight compared to the glycosylated forms, also the bands should gain sharpness.
Arndt Rohwedder
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