Preparation of a neoglycoprotein using a homobifunctional reagent and its applicability for protein blotting and electron microscopy.
A new method for the preparation of a neoglycoprotein (chemically mannosylated bovine serum albumin, D-Man.BSA) is described using the homobifunctional reagent divinylsulphone.D-Man.BSA purified by affinity chromatography on ConA-Sepharose 4B shows microheterogeneity as demonstrated by immunoaffinity electrophoresis with free ConA in the first-dimension gel. The dissociation constant K for the neoglycoprotein-ConA complex has been calculated to be 2.5.10(-5) M. Biotinylated D-Man.BSA is a useful reagent to detect carbohydrate binding proteins of L1210 leukemia cells on blots. The neoglycoprotein labelled with colloidal gold may be used to demonstrate L1210 cell surface D-Man binding proteins by preembedding electron microscopy.
Walzel
H
H
Department of Biochemistry, Rostock University.
Bremer
H
H
Neels
P
P
Jonas
L
L
Brock
J
J
D000818:Animals; D001710:Biotin; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000378:metabolism; D006046:Gold; D007122:Immunoelectrophoresis / Q000379:methods*; D007939:Leukemia L1210 / Q000378:metabolism; D008358:Mannose / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods*; D009363:Neoplasm Proteins / Q000032:analysis; D012709:Serum Albumin; D012710:Serum Albumin, Bovine / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D013450:Sulfones
D008565:Membrane Proteins; D009363:Neoplasm Proteins; D012709:Serum Albumin; D013450:Sulfones; C023840:mannose-bovine serum albumin conjugate; D003208:Concanavalin A; D012710:Serum Albumin, Bovine; C009873:divinyl sulfone; D001710:Biotin; D006046:Gold; D008358:Mannose