Immunoprecipitation/deglycosylation/western blot?

Neal Robert Melvin nrmelvin at ucalgary.ca
Sat Mar 18 04:28:51 EST 2000


I'm tryin to deglycosylate a membrane
protein, which I then plan to examine on a western blot to determine what
proportion of its molecular weight is due to N-linked oligos. I have tried this with a large amount of
PNGase F for a variety of times (up to 3 days at 37 degress), but this
fails to deglycosylate my antigen. I had read a paper where they briefly
mention a protocol for deglycosylating this protein, in which the authors
first immunoprecipitate, then deglycosylate this enriched fraction. My
question is: Can I do the deglycosylation in the presence of my protein A
beads? Or is it best to release the antigen/antibody complex via boiling,
spin the beads out, and then run my deglycosylation on the supernatant?  

Neal Melvin
University of Calgary - Faculty of Medicine
Department of Neuroscience - Neuroscience Research Group (NRG)
Health Sciences Centre
3330 Hospital Drive, NW
Calgary, Alberta, Canada
T2N 4N1

e-mail: nrmelvin at ucalgary.ca
Phone: (403) 220-4490 (lab)
       (403) 220-7035 (office)
       (403) 283-8731 (fax)
	









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