Recombinant Human PANK1 His-tag Protein, CF Summary
Lys231-Lys598, with a N-terminal Met & 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 and TCEP.|
|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.
- Universal Kinase Activity Kit (Catalog # EA004)
- 1X Assay Buffer: 25 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 10 mM CaCl2, pH 7.0 (10X Assay Buffer in kit EA004)
- Recombinant Human PANK1 (rhPANK1) (Catalog # 11161-PK)
- Pantothenic Acid (Sigma, Catalog # P5155), 20 mM stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare 1X Assay Buffer by diluting 10X stock (Phosphatase Buffer 4 that is provided in kit) 10 fold with deionized water.
- Prepare a standard curve from the 1 mM Phosphate Standard (provided in kit) by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X 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 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Prepare a Reaction Mixture containing 0.4 mM ATP and 2 mM Pantothenic Acid in 1X Assay Buffer.
- Dilute Coupling Phosphatase 4 to 10 µg/mL in 1X Assay Buffer.
- Dilute rhPANK1 to 33.34 µg/mL in 1X Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
- Load 15 µL of the 33.34 µg/mL rhPANK1 into empty wells of the same plate as the curve. 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.
- Start the reaction by adding 25 µL of Reaction Mixture to all wells, excluding standard curve.
- Seal and incubate 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.
**Under these conditions, the coupling rate is 0.475. Per Reaction:
- rhPANK1: 0.5 µg
- Coupling Phosphatase 4: 0.1 µg
- ATP: 0.2 mM
- Pantothenic Acid: 1 mM
The pantothenate kinase (PANK) gene family contains four members and encodes five functional isoforms. The PANK enzymes are key to the synthesis of coenzyme A (CoA) (1,2). CoA is used by around 4% of intracellular enzymes as a co-substrate. PANK1 is the key regulatory enzyme in the CoA biosynthetic pathway and plays similar roles in both bacterial and mammalian cells (3). PANK1 expression levels are high in liver, kidney, and heart tissues (4). Mutations in PANK1 have been linked to Hallervorden-Spatz syndrome, an autosomal recessive neurodegenerative disorder caused by excessive brain iron accumulation due to the defects in CoA synthesis (5). The activity of this enzyme has been measured with a phosphatase-coupled method (6).
- Zhou, B. et al. (2001) Nat Genet. 28:345.
- Polsterm, B.J. et al. (2010) Gene 465:53.
- Robishaw, J.D. et al. (1985) Am. J. Physiol. 1:248.
- Rock, C.O. et al. (2000). J Biol Chem. 275:1377.
- Gordon, N. (2002) Eur J Paediatr Neurol. 6:243.
- Wu, Z.L. (2011) PLoS ONE 6:e23172.
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