Recombinant E. coli N-Acetyl-D-Glucosamine Kinase/NAGK, CF Summary
Met1-Asp303 with C-terminal 6-His tag
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, Glycerol, Brij-35 and DTT.|
|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: 25 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 10 mM CaCl2, pH 7.0
- Recombinant E.coli N‑Acetyl‑D‑Glucosamine Kinase/NAGK (rE.coli NAGK) (Catalog # 8020-GK)
- Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water
- N-acetyl-alpha -D-glucosamine (GlcNAc) (Calbiochem, Catalog # 1079), 1 M stock in deionized water
- Universal Kinase Activity Kit (Catalog # EA004)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- 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 Assay Buffer for a 100 µM stock.
- Prepare 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 nmol per well.
- Prepare a reaction mixture composed of 0.5 mM ATP and 12.5 mM GlcNAc in Assay Buffer.
- Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in Assay Buffer.
- Dilute rE.coli NAGK to 1 µg/mL in Assay Buffer.
- 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 the 1 µg/mL rE.coli NAGK into the plate. Include a control containing 20 µL of Assay Buffer.
- Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to the wells, excluding the standard curve.
- Add 20 µL of reaction mixture to the wells, excluding the standard curve.
- Cover the plate with a plate sealer and incubate 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.
** Under these conditions, the coupling rate is 0.475.
- rE.coli NAGK: 0.020 µg
- Coupling Phosphatase 4: 0.1 ug
- ATP: 0.2 mM
- GlcNAc: 5 mM
Background: N-Acetyl-D-Glucosamine Kinase/NAGK
N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) are repeating sugar units of peptidoglycan, the major component of bacterial cell wall structure and a drug target of various antibiotics including penicillin (1). Recently, interest has been generated regarding cell wall peptidoglycan catabolism, because as much as 50% of the peptidoglycan is turned over in one generation of bacterial growth (2). N-acetylglycosamine kinase (nagK) is a key enzyme for the recycling of GlcNAc in E. coli (3). Due to its high activity, it can be used for efficient conversion of GlcNAc to GlcNAc-6-phosphate. The enzyme is assayed using a phosphatase-coupled kinase assay (4).
- Plumbridge, J. (2009). J. Bacteriol. 191:5641.
- Reith, J. et al (2011). J. Bacteriol. 193:5386.
- Uehara, T. and Park, J.T.(2011) J. Bacteriol. 186:7273.
- Wu, Z.L. (2011) PLoS One 6:e23172.
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