Recombinant Human GCNT2 His-tag Protein, CF Summary
0.25 μg of Recombinant Human GCNT2 will convert >50% of its substrate to product, as measured under the described conditions.
Asn26-Phe400, with a C-terminal 6-His
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 50 mM MES, 1 mM DTT, pH 6.5
- Recombinant Human GCNT2 (rhGCNT2) (Catalog # 10847-GT)
- Cy5-labeled Extended G2 (Catalog # GL303)
- Recombinant Human B4GalT1 (rhB4GALT1) (Catalog # 3609-GT)
- UDP-Gal (Sigma, Catalog # U4500), 10 mM stock in Deionized Water
- UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol/50% Deionized Water
15% SDS-PAGE Gel
- Reducing SDS-PAGE gel loading buffer
- Fluorescent imager
- Dilute rhGCNT2 to 25 µg/mL in Assay Buffer.
- Prepare a Master Mix containing 0.2 µM Cy5-labeled Extended G2, 1 mM UDP-GlcNAc, 1 mM UDP-Gal and 50 µg/mL rhB4GALT1 in Assay Buffer.
- Prepare reaction(s) by combining 10 µL of 25 µg/mL rhGCNT2 and 10 µL of Master Mix. Prepare a negative control by combining 10 µL of Assay Buffer and 10 µL of Master Mix.
- Incubate the reaction(s) and control at 37 °C for 90 minutes.
- Add 7 µL of reducing SDS-PAGE gel loading buffer to each reaction and control. Mix.
- Load 13.5 µL of each reaction and control per lane on a 15% SDS-PAGE gel. Run dye front down 80% of the length of the gel (minimum).
- Visualize the gel with a fluorescent imager.
- Calculate percent conversion of the substrate to product.
- rhGCNT2: 0.25 µg
- Cy5-labeled Extended G2: 2 pmol
- UDP-Gal: 0.5 mM
- UDP-GlcNAc: 0.5 mM
- rhB4GALT1: 0.5 µg
GCNT2 (Catalog # 10847-GT) recognizes polylactosamine structure and can tolerate core-6 fucose and its Cy5 derivatives.
1 μg/lane of Recombinant Human GCNT2 His-tag (Catalog # 10847-GT) was resolved with SDS-PAGE under reducing (R) conditions and visualized by silver staining, showing bands at 53-63 kDa.
Lane 1 contained substrate glycan extended G2 (GL303). In the presence of rhGCNT2, the glycan was modified, and a mobility shift was observed.
Background: Glucosaminyl (N-acetyl) Transferase 2/GCNT2
N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase (GCNT2) is a key branching enzyme that converts linear into branched poly-N-acetyllactosaminoglycans. It is responsible for the formation of the blood group I antigens during embryonic development and is closely associated with the development and maturation of erythroid cells (1, 2, 3). GCNT2 is a Golgi resident type II membrane protein with a typical domain structure of glycosyltransferases. GCNT2 has been found to be downregulated in melanomas, which leads to the loss of N-linked I-branched glycans and the synthesis of poly-N-acetyllactosamine (i-linear) glycans (4). In addition, GCNT2 has been found to play a role in breast cancer and lung cancer and knockdown of GCNT2 expression decreased cell migration and invasion in vitro (5). The activity of recombinant GCNT2 is demonstrated in an electrophoretic gel mobility shift assay using a fluorophore-labeled glycan as the substrate (6).
- Marti, F.A. et al. (1995) Glycobiology 5:417.
- Bierhuizen, M.F.A. et al. (1993) Genes Dev. 7:468.
- Inaba, N. et al. (2003) blood 101:2870.
- Sweeney, Jenna Geddes et al. (2018) Nature communications 9:3368.
- Haijun, Zhang et al. (2011) Cancer Res. 71:4846.
- Wu, Z.L. et al (2020) Glycobiology 30:970.
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