Recombinant Human LARGE1 His-tag Protein, CF Summary
- R&D Systems CHO-derived Recombinant Human LARGE1 His-tag Protein (11634-LG)
- Quality control testing to verify active proteins with lot specific assays by in-house scientists
- All R&D Systems proteins are covered with a 100% guarantee
Product Specifications
Phe34-Ser756, with an N-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
11634-LG
| 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 Procedure
- Assay Buffer: 100 mM Sodium Acetate, 10 mM MnCl2, pH 5.5
- Recombinant Human LARGE1 (rhLARGE1) (Catalog # 11634-LG)
- Donor Substrate: UDP-Xylose, 10 mM stock in deionized water
- Acceptor Substrate: 4-Methylumbelliferyl-beta -D-Glucuronide (4-MUG), 50 mM stock in DMSO
- Glycosyltransferase Activity Kit (Catalog # EA001)
- Clear 96-well Plate
- Plate Reader with Absorbance Read Capability
- Dilute UDP-Xylose to 0.3125 mM in Assay Buffer.
- Dilute Coupling Phosphatase 1 (supplied in kit) to 10 µg/mL in Assay Buffer.
- Dilute 4-MUG to 10 mM in Assay Buffer.
- Prepare Reaction Mixture by combining equal volumes of 0.3125 mM UDP-Xylose, 10 µg/mL Coupling Phosphatase 1 and 10 mM 4‑MUG.
- Dilute rhLARGE1 to 37.5 µg/mL in Assay Buffer.
- Dilute 1 mM Phosphate Standard (supplied in kit) by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
- Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5.0 nmol per well.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 20 µL of 50 µg/mL rhLARGE1 into the plate. Include a Control containing 20 µL of Assay Buffer.
- Start the reaction by adding 30 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
- Seal plate and incubate at 37 °C for 3 hours.
- Add 30 µL of Malachite Green Reagent A (supplied in kit) to all wells. Mix briefly.
- Add 100 µL of deionized water to all wells. Mix briefly.
- Add 30 µL of Malachite Green Reagent B (supplied in kit) to all wells. Mix briefly and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Phosphate released* (nmol) x (1000 pmol/nmol) |
| Incubation time (min) x amount of enzyme (µg) |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
- rhLARGE1: 0.75 µg
- Coupling Phosphatase 1: 0.1 µg
- UDP-Xylose: 0.0625 mM
- 4-MUG: 2 mM
Scientific Data
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Recombinant Human LARGE1 His-tag (Catalog # 11634-LG) is responsible for addition of alternating of xylose and glucuronic acid repeating units to form polysaccharide matriglycan.
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Recombinant Human LARGE/N-His Protein (Catalog # 11634-LG-Develeopment) has a molecular weight (MW) of 182.3 kDa as analyzed by SEC-MALS, suggesting that this protein is a dimer. MW may differ from predicted MW due to post-translational modifications (PTMs) present (i.e. Glycosylation).
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2 μg/lane of Recombinant Human LARGE1 His-tag (Catalog # 11634-LG) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 86-95 kDa, under reducing conditions.
Reconstitution Calculator
Background: LARGE1
- Katz, M., et al. (2022) PLoS One. 17:e0278713.
- Yoshida-Moriguchi T and KP Campbell. (2015) Glycobiology. 25:702.
- Fujimura, K. et al. (2005) Biochem. Biophys. Res. Commun. 329:1162.
- Longman, C. et al. (2003) Hum. Mol. Genet. 12:2853.
- Clement, E. et al. (2008) Ann. Neurol. 64:573.
- Meilleur, K.G. et al. (2014) J. Neuropathol. Exp. Neurol. 73:425.
- De Bernabe, D.B. et al. (2009) J. Biol. Chem. 284:11279.
- Liu, Y., et al. (2021) Pharmgenomics Pers. Med. 14:87.
- Barresi, R. et al. (2004) Nat. Med. 10:696.
- Godfrey, C. et al. (2011) Curr. Opin. Genet. Dev. 21:278.
- Hewitt, J.E. (2012) Genome Med. 4:23.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
FAQs
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