Recombinant Human PLA2G4A Protein, CF
Recombinant Human PLA2G4A Protein, CF Summary
Met1-Ala749, with a 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 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, 0.5 M NaCl, 20 mM CaCl2, 2 mg/mL BSA, pH 8.5. Note: Do not vortex, mix by gentle inversion.
- Enzyme Buffer: 50 mM Tris, 0.5 M NaCl, 2 mg/mL BSA, pH 8.5. Note: Do not vortex, mix by gentle inversion.
- Recombinant Human PLA2G4A (rhPLA2G4A) (Catalog # 6659-PL)
- Substrate: 1-Hexadecanoyl-2-(1-Pyrenedecanoyl)-sn-Glycero-3-Phosphocholine (Invitrogen, Catalog # H361), Dilute to 2 mM in DMSO, then dilute to a final stock concentration of 400 µM in ethanol
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Thaw Substrate at 37 °C for five minutes. Mix well by vortexing the warmed solution for 30 seconds.
- Dilute rhPLA2G4A to 2 µg/mL in Enzyme Buffer. Do not vortex, mix by gently inverting the tube 5 times.
- Dilute Substrate to 10 µM in Assay Buffer. Do not vortex, mix by gently inverting the tube 5 times.
- Load 50 µL of the 2 µg/mL rhPLA2G4A into a black well plate, and start the reaction by adding 50 µL of 10µM Substrate. Include a Substrate Blank containing 50 µL Enzyme Buffer and 50 µL of 10 µM Substrate without any rhPLA2G4A. Mix the wells by pipetting up and down 3 times.
- Read at excitation and emission wavelengths of 345 nm and 395 nm (top read), respectively, in kinetic mode for 5 minutes immediately after mixing the Substrate and enzyme.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard 1-pyrenedecanoic acid (Invitrogen, Catalog # P31).
- rhPLA2G4A: 0.1 µg
- Substrate: 5 µM
Cytosolic Phospholipase A2a (PLA2G4A) belongs to the group IV phospholipase A2 family (1). These enzymes hydrolyze the ester bond at the second position (sn-2) of membrane glycerophospholipids. The group IV phospholipase A2 (PLA2G4) family is comprised of six intracellular enzymes (2). PLA2G4A has a preference for arachidonic acid in the sn-2 position of phospholipids indicating its involvement in the metabolism of eicosanoids. Its active site is characterized by a serine and aspartic acid dyad. The overall structural feature displays two domains, a lipid binding C2 domain and a catalytic a/b hydrolase domain. PLA2G4A translocation to the membrane and activation is facilitated by a calcium‑ion dependent conformational change in its C2 domain. In addition, phosphorylation of serine residues in PLA2G4A by protein kinases may be another regulatory mechanism for promoting the release of arachidonic acid. The knockout mouse studies of this enzyme indicate that the enzyme may play a major role in inflammatory diseases. Genetic ablation or reduction of PLA2G4A protected human amyloid precursor protein (APP) related pathogenesis from a mouse model (3). Mouse model studies of brain and lung cancer indicates that PLA2G4A may play a key regulatory role in angiogenesis of these tumors (4).
- Burke, J. E. and E.A. Dennis (2009) J. Lipid Res. 50:S237.
- Ghosh, M. et al. (2006) Prog. Lipid Res. 45:487.
- Sanchez-Mejia, R.O. et al. (2008) Nat. Neuroscience 11:1311.
- Linkous, A.G. et al. (2010) J. Natl. Cancer Inst. 102:1398.
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