Recombinant Mouse IMPAD1 Protein, CF Summary
Glu51-His356 with an N-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 and Glycerol.|
|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: 50 mM Tris, 15 mM MgCl2, pH 7.5
- Recombinant Mouse Inositol Monophosphatase 3/IMPAD1 (rmIMPAD1) (Catalog # 7028-PD)
- Adenosine 3′,5′-diphosphate (PAP) (Sigma, Catalog # A5763), 20 mM stock in deionized water
- 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 PAP to 1 mM in Assay Buffer.
- Dilute rmIMPAD1 to 1 µg/mL in Assay Buffer.
- 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. 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 Assay Buffer. The standard curve has a range of 0.078 to 5.0 nmol per well.
- 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 1 µg/mL rmIMPAD1 into the plate. Include a Control containing 25 µL of Assay Buffer.
- Start the reaction by adding 25 µL of 1 mM PAP to the wells, excluding the standard curve and curve blank.
- Cover the plate with parafilm or 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)|
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.Per Reaction:
- rmIMPAD1: 0.025 μg
- PAP: 0.5 mM
Background: Inositol Monophosphatase 3/IMPAD1
Sulfur metabolism is critical for multicellular organism development (1). Sulfur is activated in vivo to form the sulfur donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (2), and then transferred to acceptor molecules, such as glycosaminoglycans (3), steroids and xenobiotics (4). Following transfer, PAPS is converted to 3'‑phosphoadenosine‑5'‑phosphate (PAP). PAP causes inhibition of many sulfotransferases and is therefore cytotoxic (5). IMPAD1 is a recently discovered PAP‑specific phosphatase that removes the 3'‑phosphate from PAP to form non‑toxic AMP (6), which can subsequently be recycled by cells. The enzyme is localized in the Golgi apparatus where glycosaminoglycan synthesis occurs. Mouse IMPAD1 exhibits 91% sequence identity with the human homologue. R&D Systems has found that commercial preparations of PAP may contain trace lithium salts, which is a potent micromolar-level inhibitor of IMPAD1 (6).
- Stott, C.A. (2002) Endocr. Rev. 23:703.
- Robbins, P.W. and Lipmann, F. (1956) J. Am. Chem. Soc. 24:6409.
- Plaas, A. H. et al. (1998) J. Biol. Chem. 273:12642.
- Nowell, S. and Falany, C.N. (2006) Oncogene 25:1673.
- Gamage, N. et al. (2006) Toxicol. Sci. 90:5.
- Frederick, J.P. et al. (2008) Proc. Natl. Acad. Sci. USA 105:11605.
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Phosphatase Activity Assays
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