Recombinant S. cerevisiae PPA1 Protein, CF
Recombinant S. cerevisiae PPA1 Protein, CF Summary
Thr2-Val287, with an N-terminal Met and 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, DTT and MgCl.|
|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 Buffer: 25 mM MES, 8 mM MgCl2, pH 7.0
- Recombinant Yeast Inorganic Pyrophosphatase/PPA1 (ryPPA1) (Catalog # 8088-PP)
- Substrate: Sodium Pyrophosphate (Sigma, Catalog # P8010), 125 mM stock in deionized water
- Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- 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 Assay Buffer for a 100 µM stock.
- Prepare the standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
- Dilute Substrate to 1 mM in deionized water.
- Dilute ryPPA1 to 0.01 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 25 µL of the 0.01 µg/mL ryPPA1 into the plate. Include a Substrate Blank containing 25 µL of Assay Buffer.
- Start the reaction by adding 25 µL of 1 mM Substrate to the wells, excluding the standard curve and curve blank.
- 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 2**|
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Substrate Blank.
** Note that 2 mol of phosphate are produced for every 1 mol of Pyrophosphate consumed.
- ryPPA1: 0.00025 µg
- Sodium Pyrophosphate: 0.5 mM
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).
- Harold, FM (1966) Bacteriol. Rev. 30:772.
- Nelson, D.L. and Cox, M.M. (2000) Lehninger Principles of Biochemistry, 3rd ed. pp.937. ISBN 1-57259-153-6.
- 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.
No product specific FAQs exist for this product, however you mayView all Proteins and Enzyme FAQs
Phosphatase Activity Assays
Phosphatase Peptide Substrates
Supplemental ELISA Products
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