Methods to measure amount of glycans - (Nov/26/2007 )

Hello!

I need advices on how to measure / quantify the amount of glycans. I'm basically working with 2 viruses: wild type and mutant, and I want to see if there's any glycosylation difference on the viral envelope protein. I've sent my the wild type and mutant viruses to MS-ES, but all my collaborators could tell me is that there's no qualitative difference between the two, and all I want to know is really if there's any difference in terms of quantity!

I've tried to digest my virus with pNGase alone, but whether I do a time course or a dilution of enzyme, the western blot resulted is very smeary and yields no helpful information. I've also tried trypsin and subtilising-A digestion, but again, the WB (gradient) was very smeary. Is there anything else I can do???

Also, I've to work in the context of a whole virus, so producing viral protein in CHO cells or bacular system will NOT work for me, unfortunately!

Will be greatly appreciated if anyone can provide any insight on this! MANY THANKS!

I was womdering if lectins with different sugar specificity can be use in this case, like a WB with biotinylated lectin, ConA, WGA, RCA, etc, followed by ABC.

hi chevil,

could you elaborate more what you want to quantify..?
well as such there are multiple method to quantify glycans most of them are chromatographic based..? but if you wanna have a rough estimastion you can do it with total carbohydrate quantification kit ( from sigma)..this an easy-to-use kit this may also get a rough idea regarding difference in amount of glycosylation..
hope I have been helpful…

regarding your westernblot problem (although I am not a expert on this) it may be due to the chemicals from PNGase digestion mixture..just check it up

tc

we had the glycans of our glycoprotein determined by gc-mass spectrometry. we gave our glycoprotein to another investigator and he used this method (or so he said):

Neeser, J. R. and Schweizer, T. F. (1984) Anal. Biochem. 142, 58-67.