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!
srps at galactose.mc.duke.edu
"Insert your own pithy phrase just about here!"