Detection of Biotin Labeled Glycoproteins on Western Blots

Biotin-hydrazide modification of oxidized glycans (see Figure 1.) can be utilized to label glycoproteins prior to electrophoresis or after Westernblotting.1

Carbohydrate residues in solutions of glycoproteins are chemically oxidized by incubation for 30 minutes using sodium periodate (final concentration 1mM for oxidation of terminal sialic acid residues or 10 mM for oxidation of general sugar moieties). After reaction, any excess periodate is removed by dialysis against phosphate buffered saline. The glycoprotein is subsequently incubated for 2 hours with a 5 mM solution of biocytin hydrazide or biotin hydrazide to attach biotin to the oxidized glycan sites. After reaction and dialysis, the biotin-labeled glycoprotein may be applied to an electrophoresisgel.

Glycoproteins on blots may also be labeled using biocytin hydrazide. Bayer, et al., reported that biocytin hydrazide, but not biotin hydrazide, is suitable for labeling of blotted glycoconjugates.1 The procedure is similar to that used for glycoproteins in solution. The Western blot must be blocked with an appropriate protein prior to incubation with sodium periodate solution (1 mM or 10 mM) for 30 minutes. After incubation, the blot is then rinsed thoroughly and incubated with 3 μg/mL biocytin hydrazide for 1 hour. Enzymatic oxidation of carbohydrates can be performed using neuraminidase to oxidize terminal sialic acid groups and galactose oxidase to oxidize terminal galactose groups. The enzyme solution(s) and biocytin hydrazide are both included in the reaction for simultaneous, rather than sequential, labeling. Solutions of glycoproteins (0.5-3 mg/ml) and glycoproteins immobilized on Western blots may be labeled by enzymatic means.

After electrophoresis and Western transfer, biotin-conjugated glycoproteins can be probed with streptavidin-peroxidase. Detection can be accomplished with either a colorimetric tetramethylbenzidine (TMB) or chemiluminescent peroxidase substrate. Alkaline phosphatase-conjugated streptavidin can also be used to probe the blots with subsequent detection using a colorimetric BCIP/NBT or CDP-Star® Chemiluminescent Substrate. Typical detection limits for glycoproteins using the streptavidin-peroxidase method are ~ 50-100 ng. Optimal results are obtained on PVDF membranes.

References:
1. Bayer, E.A., et al., Analysis of proteins and glycoproteins on blots. Meth. Enzymol., 184, 415-427 (1990).
2. Bayer, E.A., et al., Biocytin hydrazide--a selective label for sialic acids, galactose, and other sugars in glycoconjugates using avidin-biotin technology. Anal. Biochem., 170, 271-281 (1988).

For more information on Glycobiology, browse our online Glycobiology Analysis Manual.
Figure 1. Schematic of biotin labeling reaction to periodate oxidized glycan

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B3770 (+)-Biotinamidohexanoic acid hydrazide ≥90% (TLC), powder
B7639 (+)-Biotin hydrazide ≥97% (TLC), powder
CPS160 Chemiluminescent Peroxidase Substrate-1 sufficient for 60 mL substrate
CPS1120 Chemiluminescent Peroxidase Substrate-1 sufficient for 120 mL substrate
CPS1300 Chemiluminescent Peroxidase Substrate-1 sufficient for 300 mL substrate
CPS350 Chemiluminescent Peroxidase Substrate-3 sufficient for 50 mL substrate
CPS3500 Chemiluminescent Peroxidase Substrate-3 sufficient for 500 mL substrate
G7907 Galactose Oxidase from Dactylium dendroides 500-1,500 units/mg protein
N7885 Neuraminidase from Vibrio cholerae Type III, buffered aqueous solution, 0.2 μm filtered, 1-5 units/mg protein (Lowry, using NAN-lactose)
311448 Sodium periodate ACS reagent, ≥99.8%
S1878 Sodium (meta)periodate ≥99.0%
S2890 Streptavidin−Alkaline Phosphatase from Streptomyces avidinii lyophilized powder, 700-2,300 DEA units/mg protein
S5512 Streptavidin−Peroxidase from Streptomyces avidinii lyophilized powder
T0565 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System for Membranes ready to use solution