Recombinant Human PLA2G4A Protein, CF

Catalog # Availability Size / Price Qty
6659-PL-010
Product Details
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Recombinant Human PLA2G4A Protein, CF Summary

Product Specifications

Purity
>80%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to hydrolyze 1-Hexadecanoyl-2-(1-pyrene-decanoyl)-sn-glycero-3-phosphocholine. The specific activity is >30 pmol/min/μg, as measured under the described conditions.
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human PLA2G4A protein
Met1-Ala749, with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Analysis
Ser2
Predicted Molecular Mass
86 kDa
SDS-PAGE
85-95 kDa, reducing conditions

Product Datasheets

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

6659-PL

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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • 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
  1. Thaw Substrate at 37 °C for five minutes. Mix well by vortexing the warmed solution for 30 seconds.
  2. Dilute rhPLA2G4A to 2 µg/mL in Enzyme Buffer. Do not vortex, mix by gently inverting the tube 5 times.
  3. Dilute Substrate to 10 µM in Assay Buffer. Do not vortex, mix by gently inverting the tube 5 times.
  4. Load 50 µL of the 2 µg/mL rhPLA2G4A into a black well plate, and start the reaction by adding 50 µL of 10µM 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.
  5. 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.
  6. 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).

Per Well:
  • rhPLA2G4A: 0.1 µg
  • Substrate: 5 µM
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: PLA2G4A

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).

References
  1. Burke, J. E. and E.A. Dennis (2009) J. Lipid Res. 50:S237.
  2. Ghosh, M. et al. (2006) Prog. Lipid Res. 45:487.
  3. Sanchez-Mejia, R.O. et al. (2008) Nat. Neuroscience 11:1311.
  4. Linkous, A.G. et al. (2010) J. Natl. Cancer Inst. 102:1398.

Long Name
Phospholipase A2 Group IV A
Entrez Gene IDs
5321 (Human); 18783 (Mouse); 24653 (Rat)
Alternate Names
calcium-dependent phospholipid-binding protein; cPLA2; cPLA2-alpha; lysophospholipase; MGC126350; phosphatidylcholine 2-acylhydrolase; Phospholipase A2 group IVA; phospholipase A2, group IVA (cytosolic, calcium-dependent); PLA2G4A; PLA2G4cytosolic phospholipase A2

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