Glycoprotein sequencing & PVDF membrane transfer - (Sep/14/2006 )

After purifying my glycoprotein, the next step is to find its amino acid sequence. Is it necessary to deglycosilate it first? If so, which is the best method (simple and cheap!) to do it?

By the way, I've heard that we can run an electrophoresis and do the transfer to the PVDF membrane, from which we can take the protein to the amino acid sequence analysis (Edman analysis). Can nitrocellulose be used as well? [font=Comic Sans Ms][size=2]

Any other useful tips?

PVDF has better binding properties and is better to handle than nitrocellulose; some as Millipore provide special PVDF (immobilon PQ) for optimum sequence performance; deglycosilation of N- or O-glycosides can be done with appropriate glycosidases even on blots but may contaminate your sample with protein (glycosidases)

nitrocellulose will react with the chemicals used by the sequencer. pvdf is the only way to go for this purpose. you can also stain the blot with coomassie prior to sequencing.

i have tried to sequence a glycoprotein and found that it was n-terminally blocked. i don't know if this is because of glycosylation but may be. you can remove virtually all glycoside linkages by sequential digestion with neuraminidase, o-glycanase and n-glycanase (these are trade names for glycosidases but i forget their generic names). the n-glycanase (if not all three) can be used on sds denatured protein.

de-glycosylation is preffered for sequence analysis. also, you're probably better off using LS-MS/MS than edman sequencing for this. you'll get more data that way, and you'll probably need much less starting material. additionally, you can probably get around blotting to PVDF, and simply just cut a stained band from your SDS-PAGE gel.

After purifying my glycoprotein, the next step is to find its amino acid sequence. Is it necessary to deglycosilate it first? If so, which is the best method (simple and cheap!) to do it?

By the way, I've heard that we can run an electrophoresis and do the transfer to the PVDF membrane, from which we can take the protein to the amino acid sequence analysis (Edman analysis). Can nitrocellulose be used as well? [font=Comic Sans Ms][size=2]

Any other useful tips?