Enzyme assay of beta-1,4-N-acetylglucosaminyltransferase-IV (GnT-IV)

 Dissolve 2.5 g human apo-transferrin in 100 mL of 50 mM Tris-HCl (pH7.2) containing 1% (v/v) β-mercaptoethanol and 2.5% (w/v) SDS.

 Mix gently with stirring on a magnetic stirrer, and incubate at 60˚C for 2 h.

 After cooling to room temperature, add 4.0% (w/v) of Nonidet P-40 and 75 U of N-glycosidase F. Add 100 μL of toluene to prevent bacterial growth. Incubate at 37˚C overnight.

 Add an additional 75 U of N-glycosidase F, incubate at 37˚C overnight. Analyze the aliquot of sample by SDS-PAGE.

 Divide into 20 mL aliquots in 50 mL conical tubes. Add equal volume of TE-saturated phenol/chloroform solution and mix vigorously by vortex.

 Centrifuge at 4,000 rpm for 20 min.

 Transfer the clear aqueous phase to a bottle and lyophilize.

 Dissolve the lyophilized powder in 20 mL of 50 mM ammonium acetate buffer (no pH adjustment).

 Apply the sample to a Bio-gel P-100 column (2.5 cm × 70 cm) equilibrated with 50 mM ammonium acetate buffer at the flow-rate of 8 drops/sec. Collect the eluate at 2mL/tube.

 Detect sugar chains by phenol/H₂SO₄ method. The sugar chains will be eluted between fractions 130 and 170. Transfer the carbohydrate-positive fractions to a 500 mL bottle and lyophilize.

 Prepare PA reagent. Dissolve 27 g 2-aminopyridine in 13 mL of glacial acetic acid.

 Add the PA reagent to the lyophilized sugar chains and stir on a magnetic stirrer until completely dissolved. Wrap the bottle in aluminum foil. Incubate at 90˚C for 1 h by bath.

 Prepare Reducing reagent. Dissolve 57 g borane-dimethylamine complex and 23 mL of glacial acetic acid in 14mL of water.

 Add the reducing reagent to the bottle and stir on a magnetic stirrer. Incubate at 80˚C for 1 h in a fume hood.

 After cooling to room temperature, adjust volume to 250 mL with H₂O.

 Transfer the sample to a separatory funnel. To remove the excess 2-aminopyridine, extract the sample twice with equal volumes of TE-saturated phenol/chloroform, and once with chloroform. The top aqueous phase contains the PA-labeled N-glycans.

 Concentrate the sample to 40 mL by evaporation with a rotary evaporator.

 Apply the concentrated sample to a HW-40F column (5 cm × 25 cm) equilibrated with 10 mM ammonium acetate buffer (pH 6.0) at the flow-rate of 2.5 mL/min. Collect the eluate at 5mL/tube.

 Detect sugar chains by phenol/H₂SO₄ method. The absorption of eluted PA can be monitored at 300 nm. The PA-labeled N-glycans will be eluted between fractions 40 and 70. Collect the carbohydrate-positive fractions and lyophilize. Analyze the yield of PA-labeled N-glycans by phenol/H₂SO₄ method and HPLC.

 Dissolve the lyophilized powder in 10 mL of 0.4 M sodium acetate buffer (pH 5.0) containing 4 mM MgCl₂.

 Add 10 U of neuraminidase and 10 mg of β-galactosidase. Add one drop of toluene to prevent bacterial growth. Incubate at 37˚C overnight.

 Analyze the aliquots of the sample by HPLC.

 Purify PA-GnGnbi by reverse-phase HPLC as following condition:

column, Vydac 218TP152010 (10 mm × 250 mm); eluent, 100 mM ammonium acetate (pH 4.0) containing 0.15% n-butanol; flow rate, column temperature, 50˚C; 2.5 mL/min; detection, 300 nm.

 Confirm the purity of PA-GnGnbi by HPLC. From 2.5 g of human apo-transferrin, 25–30 μmol of PA-GnGnbi was obtained at yield of 43 %.

 Prepare 2× GnT-IV reaction buffer: 250 mM MOPS-KOH buffer, 40 mM UDP-GlcNAc, 400 mM GlcNAc, 1% (w/v) Triton X-100, 20 % (v/v) glycerol, 1% (w/v) BSA, 15 mM MnCl₂, pH 7.3. Store −20˚C until use.

 Combine the following components in a thin-walled PCR tube.

2× GnT-IV reaction buffer 5 μL

4 mM PA-GnGnbi 2 μL

Enzyme solution 3 μL

(Total volume 10 μL)

 Mix gently by pipetting, and incubate at 37˚C for 2 h.

 Add 30 μL of 10 mM EDTA (pH 7.0), vortex and boil for 2 min.

 Centrifuge at 15,000 rpm for 15min.

 Collect the supernatant, and analyze by HPLC.

 Prepare 10 × stock (1 M ammonium acetate, pH 4.0). Dissolve 7.7 g ammonium acetate in 900 mL of Milli-Q water, and add 52 mL of glacial acetic acid. Titrate with ammonia water to pH 4.0, and add Milli-Q water to make a final volume of 1 L. Store at room temperature.

 HPLC Buffer (50 mM ammonium acetate, 0.15% n-butanol, pH 4.0). Prepare 1 L of buffer by mixing 50 mL of 10 × stock buffer, 1.5 mL n-butanol, and Milli-Q water. Filtrate the buffer through a 0.45 μm filter prior to use.

 Analyze 10 μL of the reaction mixture as following condition: column, ODS-80Tm (4.6 mm × 150 mm); eluent, 50 mM ammonium acetate (pH 4.0) containing 0.15% n-butanol; flow-rate, 1.2 mL/min column temperature, 50˚C; detection, Ex = 320 nm, Em = 400 nm