Cy5-Fuc Labeled M1N1f Summary
Learn more about Fluorescent Glycan Labeling and Detection
Supplied in 20 mM Tris, pH 8.0
Stability & Storage
Store at < -20 °C. Good for 6 months from date of receipt.
- Used as a ligand for lectins and to study glycan protein interaction.
- Used as a substrate for various glycosidases and glycosyltransferases.
- Used as a scaffold for the synthesis of various glycan epitopes such as sialyl Lewis X structures.
Key Features and Benefits:
- Excitation at 649 nm and emission at 671 nm.
- The fluorescent dye Cy5 is conjugated to the C6 position of the core-6 fucose.
- Can be separated on 15%-17% SDS-PAGE and directly visualized as a single band under a fluorescent imager.
- Linear response range for Cy5-labeled glycans can be from 10 fmol to 100 pmol, depending on the sensitivity of detection.
- Store at < -20 °C. Good for 6 months from date of receipt.
- Wu et al., (2019) Glycobiology 29:750-754
- Wu et al., (2020) Glycobiology 30:970-980
- A Simple SDS-PAGE-based Technique for Probing Terminal Glycans
- GDP-Cy5-Fucose (ES301)
- CMP-Cy5-Sialic Acid (ES302)
- GDP-Cy3-Fucose (ES401)
- CMP-Cy3-Sialic Acid (ES402)
Enzymes and detection reagents
Cy5-Fuc Labeled M1N1f as a substrate for recombinant MGAT2 assay. Cy5-Fuc labeled M1N1f (M1N1f) (Catalog # GL301) (0.2 pmol per reaction) was treated by a serial dilution of Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2 Protein (Catalog # 10711-GT). The reactions were incubated 30 minutes at 37oC then heat inactivated, and further elongated with Recombinant Human B4GalT1 Protein (Catalog # 3609-GT) at 37oC for 20 minutes. Samples were separated on 15% SDS-PAGE and imaged with TCE staining (top panel) and fluorescent imaging (lower panel). Product 2 is visible with 6.25 ng of MGAT2, and its maximal level was achieved at 100 ng of the enzyme. The conversion of M1N1f to M2f by MGAT2 did not generate obvious mobility shift (data not shown). To overcome this issue, the substrate M1N1f and the product M2f were elongated by B4GalT1 to Product 1 and Product 2, respectively, to create an obvious gel mobility shift.
Complete Your Research
Cy5-Fuc Labeled M1N1f' This substrate can be used as ligand for lectins and to study various glycosidases and glycosyltransferases. This can also be used as a scaffold for the synthesis of various glycan epitopes such as sialyl Lewis X structures. The fluorescent dye Cy5 is conjugated to the C6 position of the core-6 fucose.
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.
- 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)
- 15% SDS-PAGE
- 6X gel loading dye
- A fluorescent imager
- Dilute rhMGAT2 to 10 ng/µL in the Assay Buffer.
- Create a Reaction Mix #1 by combining 0.02 µM M1N1f and 1.5 mM UDP-GlcNAc in Assay Buffer.
- Mix 10 µL of the diluted rhMGAT2 and 10 µL of the Reaction Mix #1 in a centrifuge tube.
- Prepare a negative control by mixing 10 µL of Assay Buffer and 10 µL of Reaction Mix #1 in another centrifuge tube.
- Incubate the reaction and control at 37o C for 30 minutes.
- Heat tubes to 90o C for 2 minutes, cool on ice, and quickly spin down condensation.
- Create Reaction Mix #2 by combining 50 ng/µL rhB4GALT1 and 1.5 mM UDP-Gal in Assay Buffer.
- Add 10 µL of Reaction Mix #2 to each tube.
- Incubate at 37o C for 20 minutes.
- Stop the reactions and controls by adding 6 μL of 6X gel loading dye to each tube.
- 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.
- 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
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