B4GALT1 Protein, Human (HEK293, Y285L, His)

B4GALT1 Protein, Human (HEK293 His) is responsible for the overcoming multidrug resistance in human leukemia therapy via regulating the activity of the hedgehog signaling pathway^[1][2].

B4GALT1 expression is identified as an independent adverse prognostic factor for survival in non-metastatic clear cell renal cell carcinoma modesl^[1]. B4GALT1 gene plays important roles in physiological process and disease development. B4GALT1 is shown to be responsible for the synthesis of complex-type N-linked oligosaccharides in many glycoproteins as well as the carbohydrate moieties of glycolipids ^[2]. B4GALT1 is a new candidate to maintain the Stemness of lung Cancer stem cells^[3].

Measured by its ability to transfer N-Acetyl-D-galactosamine from UDP-GalNAc to N-Acetyl-D-glucosamine that incubate at 37°C for 20 min. The specific activity is >400 pmol/min/μg.

Materials
Assay buffer: 50 mM HEPES, 10 mM MnCl₂, pH 7.5
B4GALT1 Protein, Human (HEK293, Y285L, His) (HY-P7628)
Substrates: UDP-GalNAc (HY-112174), N-Acetyl-alpha-D-glucosamine (GlcNAc, HY-A0132)
Glycosyltransferase Activity Kit

Procedure
1. Dilute UDP-GalNAc to 1 mM with assay buffer.
2. Dilute Coupled Phosphatase 1 to 20 μg/mL with assay buffer.
3. Dilute GlcNAc to 200 mM with assay buffer.
4. Prepare the reaction mixture by mixing equal volumes of 1 mM UDP-GalNAc, 20 μg/mL Coupled Phosphatase 1, and 200 mM GlcNAc.
5. Dilute Human B4GalT1 to 1 μg/mL with assay buffer.
6. Add 40 μL of 1 mM phosphate standard to 360 μL of assay buffer to dilute the 1 mM phosphate standard provided in the Glycosyltransferase Activity Kit to obtain a 100 μM stock solution.
7. Prepare the standard curve by performing six 1:2 serial dilutions of the 100 μM phosphate stock solution in assay buffer. The standard curve range is 0.078 to 5 nmol/well.
8. Add 50 μL of different concentrations of the standard curve dilutions to a 96-well plate; add 50 μL of assay buffer to the blank group.
9. Add 20 μL of 1 μg/mL Human B4GalT1 to the 96-well plate; add 20 μL of assay buffer to the blank group.
10. Add 30 μL of the reaction mixture to the wells, excluding the standard curve wells and blank wells.
11. Incubate at 37 °C for 15 minutes.
12. Add 30 μL of Malachite Green Reagent A to all wells and mix.
13. Add 100 μL of deionized water to all wells and mix.
14. Add 30 μL of Malachite Green Reagent B to all wells, mix, and incubate at room temperature for 20 minutes.
15. Read the plate at 620 nm (absorbance) in endpoint mode.
16. Calculate Specific Activity:

Human

HEK293

C-6*His

P15291-1 (G44-S398, Y285L)

2683  [NCBI]

Lumenal Domain

GRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQLFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS

Approximately 50-60 kDa, based on SDS-PAGE under reducing conditions, due to the glycosylation.

• 
  ≥ 95%, as determined by reducing SDS-PAGE.

Solution

1.Supplied as a 0.22 μm filter solution of 20 mM Tris-HCl, 150 mM NaCl, pH 8.0.
2.Supplied as a 0.22 μm filter solution of 20mM Tris-HCl, 150mM NaCl, 2mM EDTA, 20% Glycerol, pH 8.0.
Please refer to the lot-specific COA for specific buffer information.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.

• [1]. Huyang Xie, et al. Increased B4GALT1 expression associates with adverse outcome in patients with non-metastatic clear cell renal cell carcinoma. Oncotarget. 2016 May 31;7(22):32723-30.  [Content Brief]

  [2]. Huimin Zhou, et al. B4GALT1 gene knockdown inhibits the hedgehog pathway and reverses multidrug resistance in the human leukemia K562/adriamycin-resistant cell line. IUBMB Life. 2012 Nov;64(11):889-900.  [Content Brief]

  [3]. Claudia De Vitis, et al. B4GALT1 Is a New Candidate to Maintain the Stemness of Lung Cancer Stem Cells. J Clin Med. 2019 Nov 9;8(11):1928.  [Content Brief]