Cy5-Fuc Labeled M1N1f

Sample Assay Protocol for using Cy5-Fuc Labeled M1N1f/M1N1f as substrate for Recombinant Human N-acetylglucosaminyltransferase 2/MGAT2 assay

Protocols are guidelines. Parameters need to be optimized by end users. Suggested input of a Cy5-M1N1f in an assay separated on SDS-PAGE is from 0.01-1 pmol.

Materials

• Assay Buffer: 25 mM Tris, 10 mM CaCl2, pH 7.5
• Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2 (rhMGAT2) (Catalog # 10711-GT)
• Cy5-Fuc Labeled M1N1f (M1N1f) (Catalog # GL301)
• Recombinant Human B4GALT1 (rhB4GALT1) (Catalog # 3609-GT)
• UDP-Gal
• UDP-GlcNAc
• 15% SDS-PAGE
• 6X gel loading dye
• A fluorescent imager

Assay

1. Dilute rhMGAT2 to 10 ng/µL in the Assay Buffer.
2. Create a Reaction Mix #1 by combining 0.02 µM M1N1f and 1.5 mM UDP-GlcNAc in Assay Buffer.
3. Mix 10 µL of the diluted rhMGAT2 and 10 µL of the Reaction Mix #1 in a centrifuge tube. 
4. Prepare a negative control by mixing 10 µL of Assay Buffer and 10 µL of Reaction Mix #1 in another centrifuge tube.
5. Incubate the reaction and control at 37^o C for 30 minutes.
6. Heat tubes to 90^o C for 2 minutes, cool on ice, and quickly spin down condensation.
7. Create Reaction Mix #2 by combining 50 ng/µL rhB4GALT1 and 1.5 mM UDP-Gal in Assay Buffer.
8. Add 10 µL of Reaction Mix #2 to each tube.
9. Incubate at 37^o C for 20 minutes.
10. Stop the reactions and controls by adding 6 μL of 6X gel loading dye to each tube.
11. Load 18 µL each of the above reactions and controls per well on a 15% SDS-PAGE and run down 80% length of the gel.
12. Visualize the gel using a fluorescent imager for 10 seconds.

Final Assay Conditions Per Reaction

• rhMGAT2: 100 ng 
• Cy5-M1N1f: 0.2 pmol 
• UDP-GlcNAc: 0.5 mM
• rhB4GALT1: 500 ng     
• UDP-Gal: 0.5 mM