Detection of glycans?

Steven Pirie-Shepherd srps at galactose.mc.duke.edu
Tue Oct 17 21:41:20 EST 1995


I am going to perfomr a reductive deamination on a protein which I 
suspect has multiple O-linked glycans. I want to then separate said 
glycans from protein and chromatograph them on monoQ (I suspect that the 
glycans are sialylated.). I notice that sialic acid absorbs at 280nm , is 
this just free SA, or will SA in linkage to gal also absrob at 280? Can I 
use a simple 280nm detector to pixckj up sialo-glycans on monoQ?
	More questions. When I perform the reductive deamination, will 
this preocedure (45'C and 4M BA Borohydride for 16 hours under N2) 
destroy my protein? I dont care if it does, but I am looking for a way to 
separate glycanns from other junk in the experiment. if the protein 
remains >20kDa then I can use gel filtration. Further, I have never used 
Na borohydride, but it comes in a big metal case!!!!! What precautions 
must i take if I subject a 4M sol'n of this stuff to 45'C under N2 (I was 
going to flush 45mL reaction tubes with N2 and place all the reactants 
into a water bath at 45'C in the flushed tube, is this safe?).

Appreciate the help!

--


__________________________________________________
Steven Pirie-Shepherd		       		
srps at galactose.mc.duke.edu	        
"Insert your own pithy phrase just about here!"



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