Recombinant Human MAN1A1 His-tag Protein, CF

• Digestion Buffer: 50 mM MES, 15 mM CaCl₂, 1 mg/mL BSA, pH 6.0
• Labeling Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MnCl₂, pH 7.5
• Recombinant Human MAN1A1 (rhMAN-1A1) (Catalog # 10665-GH)
• Oligomannose-9 (Man-9) (Dextra Laboratories, Catalog # MC1131), 0.1 mg/mL stock in deionized water
• Recombinant Human MGAT1 (rhMGAT1) (Catalog # 8334-GT)
• UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol, 50% deionized water
• Recombinant Human FUT8 (rhFUT8) (Catalog # 5768-GT)
• GDP-Cy5-Fucose  (Catalog # ES301)
• 15% SDS-PAGE gel
• Reducing SDS-PAGE gel loading buffer
• Fluorescent imager

Digestion:

1. Dilute rhMAN1A1 to 20 µg/mL in Digestion buffer.
2. Dilute Man-9 to 20 µg/mL in Digestion Buffer.
3. Combine 5 µL of 20 µg/mL Man-9, 5 µL of 20 µg/mL rhMAN1A1 and 10 µL of Digestion Buffer. Include a Control containing 5 µL of 20 µg/mL Man-9 and 15 µL of Digestion Buffer.
4. Incubate at 37 °C for 2 hours.

Labeling:

1. Dilute rhMGAT1 to 100 µg/mL in Labeling Buffer.
2. Dilute UDP-GlcNAc to 1 mM in Labeling Buffer.
3. Dilute rhFUT8 to 100 µg/mL in Labeling Buffer.
4. Dilute GDP-Cy5-Fucose to 0.05 mM in Labeling Buffer.
5. Transfer 10 µL of each digestion to a new tube and add 5 µL of 100 µg/mL rhMGAT1, 5 µL of 1 mM UDP-GlcNAc, 5 µL of 100 µg/mL rhFUT8 and 5 µL of 0.05 mM GDP-Cy5-Fucose.
6. Incubate at 37 °C for 60 minutes.
7. Add 6 µL of Reducing SDS-PAGE gel loading buffer to each reaction.
8. Load 12 µL of each reaction onto a 15% SDS-PAGE gel and perform electrophoresis.
9. Analyze gel on a fluorescent imager.

Per Reaction:

•  rhMAN1A1: 0.05 µg
•  Man-9: 0.05 µg
•  rhMGAT1: 0.5 µg
•  UDP-GlcNAc: 5 nmol
•  rhFUT8: 0.5 µg
•  GDP-Cy5-Fucose: 0.25 nmol