Recombinant Human PPA1 Protein, CF Summary
Product Specifications
Met1-Asn289, with a C-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.
6557-PP
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol 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 Procedure
- Assay Buffer: 25 mM MES, 16 mM MgCl2, pH 7.0
- Recombinant Human Inorganic Pyrophosphatase/PPA1 (rhPPA1) (Catalog # 6557-PP)
- Substrate: Sodium Pyrophosphate (Sigma, Catalog # P8010)
- ]Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute Substrate to 16 mM in deionized water.
- Dilute rhPPA1 to 0.4 µg/mL in Assay Buffer.
- Combine 25 µL of 16 mM Substrate with 25 µL of 0.4 µg/mL rhPPA1. Also create a substrate blank by combining 25 µL of 16 mM substrate with 25 µL of Assay Buffer.
- Incubate reaction and blank for 10 minutes at room temperature.
- Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of deionized water for a 100 µM stock.
- Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in deionized water. The standard curve has a range of 0.078 to 5 nmol per well.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of deionized water.
- Dilute the incubated reaction and incubated blank 20-fold by adding 950 μL of deionized water to each tube.
- Load 50 µL of the diluted rhPPA1 reaction and substrate blank into the plate.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- Add 100 µL deionized water to all wells.
- 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 2* |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Substrate Blank.
**Note: 2 mol of phosphate are produced for every 1 mol of Pyrophosphate consumed.
- rhPPA1: 0.01 µg in reaction, 0.5 ng after 20-fold dilution
- Substrate: 8 mM in reaction, 0.4 mM after 20-fold dilution
Reconstitution Calculator
Background: Inorganic Pyrophosphatase/PPA1
Inorganic Pyrophosphatases are enzymes that catalyze the hydrolysis of pyrophosphate to two phosphate ions (1). This reaction is highly exergonic, and is utilized in many biochemical pathways, such as DNA synthesis (2) and bone formation (3), to render reactions effectively irreversible (4, 5). Likewise, inorganic pyrophosphatases can be coupled to unfavorable biochemical reactions in order to drive these reactions to completion, such as in the synthesis of activated sulfur donor 3’‑phosphoadenosine-5’-phosphosulfate (PAPS) (6). Two inorganic pyrophosphatases, i.e. PPA1 and PPA2, are found in humans. PPA1 is highly active on pyrophosphate and is ubiquitously expressed (7).
- Harold, FM (1966) Bacteriol. Rev. 30:772.
- Nelson, D.L. and Cox, M.M. (2000) Lehninger Principles of Biochemistry, 3rd ed. pp.937. ISBN 1-57259.
- Poole, K.E. and Reeve, J. (2005) Curr. Opin. Pharmacol. 5:612.
- Takahashi, K. et al. (2004) Biochem. Biophys. Res. Commun. 325:203.
- Terkeltaub, R.A. (2001) Am. J. Physiol., Cell Physiol. 281:C1.
- Wu, Z.L. et al. (2010) BMC Biotechnology 10:11.
- Fairchild, T.A. and Patejunas, G. (1999) Biochim. Biophys. Acta 1447:133.
Product Specific Notices
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