Recombinant Human Xylulokinase/XYLB Protein, CF Summary
Ala2-Glu536, with an N-terminal Met and 6-His tag
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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, NaCl, DTT and Glycerol.|
|Shipping||The product is shipped with dry ice or equivalent. 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 (10X): 250 mM HEPES, 1500 mM NaCl, 100 mM MgCl2, 100 mM CaCl2 pH 7.0 (supplied in kit)
- Recombinant Human Xylulokinase/XYLB (rhXYLB) (Catalog # 8267-XB)
- D-Xylulose (Sigma, Catalog #X4625), 100 mM stock in deionized water
- Universal Kinase Activity Kit (Catalog # EA004)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare 1X Assay Buffer by diluting 10X Assay Buffer with deionized water.
- Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock.
- Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmoles per well.
- Load 50 µL of each dilution of the standard curve into a plate in triplicate. Include a curve blank containing 50 μL of 1X Assay Buffer.
- Prepare Substrate Mixture composed of 0.4 mM ATP and 2 mM D-Xylulose in 1X Assay Buffer.
Dilute rhXYLB to 1.66 µg/mL in 1X Assay Buffer.
- Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in 1X Assay Buffer.
- Load 15 µL of the 1.66 µg/mL rhXYLB into empty wells of the same plate that contains the standard curve in triplicate. Include a Control containing 15 µL of 1X Assay Buffer.
- Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.
- Add 25 µL of Substrate Mixture to the wells, excluding the standard curve.
- Incubate sealed plate at room temperature for 10 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 100 µL of deionized water to all wells. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix 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) x coupling rate**|
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control
**The coupling rate is 0.475 under these conditions.
- rhXYLB: 0.025 µg
- Coupling Phosphatase 4: 0.1 µg
- ATP: 0.2 mM
- D-Xylulose: 1 mM
D-Xylulokinase (XYLB) catalyzes the phosphorylation of D-xylulose (Xu) to produce xylulose-5-phosphate (Xu5P) mainly in liver and kidney and is inhibited by 5‑deoxy‑5‑fluoro‑D‑xylulose (1). XYLB is the last enzyme in the glucuronate-xylulose pathway, and its product Xu5P interfaces with the pentose phosphate pathway. XYLB belongs to a family of enzymes that also include fucokinase, gluconokinase and glycerokinase. Recently, the metabolic pathway has re-emerged as a focus for drug discovery in cancer research as rapidly growing malignant tumor cells typically have glycolytic rates up to 200 times higher than those of their normal tissues of origin, i.e. the Warburg effect (2, 3). Metabolic enzymes, such as PKM2 (4) and phosphofructokinase 1 (5), are being explored as drug targets. Given that Xu5P is a key regulator of glucose metabolism and lipogenesis (1), XYLB could also be an attractive drug target. The kinase activity was measured using a phosphatase-coupled method (6).
- Bunker, R.D. et al. (2013) J. Biol. Chem. 288:1643.
- Gatenby, R.A. and Gillies, R.J. (2004). Nat. Rev. Cancer 4:891.
- Kim, J.W. and Dang, C.V. (2006). Cancer Res. 66:8927.
- Israelsen, W.J. et al. (2013) Cell 155:397.
- Yi, W. et al. (2012) Science 337:975.
- Wu, Z. (2011) PLoS ONE 6:e23172.
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