Recombinant Human PLA2G1B Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human PLA2G1B Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze 1-Hexadecanoyl-2-(1-pyrene-decanoyl)-sn-glycero-3-phosphoglycerol. The specific activity is >2,500 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human PLA2G1B protein
  Ala23-Ser148, with a C-terminal 6-His tag
• Accession #: NP_000919
• N-terminal Sequence: Ala23
• Protein/Peptide Type: Recombinant Enzymes
• Gene: PLA2G1B
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 15 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 18 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Sodium Acetate and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, 1 mg/mL BSA, pH 7.0.
  • Substrate Buffer: 50 mM HEPES, 20 mM CaCl₂, 1 mg/mL BSA, pH 7.0.
  • Recombinant Human Phospholipase A2 Group IB/PLA2G1B (rhPLA2G1B) (Catalog # 5018-PL)
  • Substrate: 1-hexadecanoyl-2-(1-pyrene-decanoyl)-sn-glycero-3-phosphoglycerol Ammonium Salt, 2 mM stock in DMSO
  • Black 96-well Plate
  • Fluorescent Plate Reader
  1. Thaw Substrate at 37 °C for five minutes. Mix well.
  2. Dilute rhPLA2G1B to 0.02 ng/µL in Assay Buffer.
  3. Dilute Substrate to 100 µM in Substrate Buffer.
  4. Load into plate, 50 µL of 0.02 ng/µL rhPLA2G1B, and start reaction by adding 50 µL of 100 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.
  5. Read at excitation and emission wavelengths of 345 nm and 395 nm (top read), respectively in kinetic mode for 5 minutes. Shake the plate for 3 seconds between each read.
  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.

  Per Well:
  • rhPLA2G1B: 0.001 µg
  • Substrate: 50 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human PLA2G1B Protein, CF

• Group IB phospholipase A2
• Phosphatidylcholine 2-acylhydrolase 1B
• phospholipase A2
• phospholipase A2, group IB (pancreas)
• PLA2
• PLA2A
• PLA2AMGC119834
• PLA2EC 3.1.1.4
• PLA2G1B
• PPLA2
• PPLA2MGC119835

Background

Secretory Phopholipase A₂ is an enzyme that hydrolyses the sn-2 ester bond of phospholipids and cell membranes, generating non-esterified free fatty acids and lysophospholipids (1‑3). Most secretory PLA₂s are stored in cytoplasmic granules and are released in the extracellular environment on appropriate cell activation. Thus, they are present at higher concentration in the plasma and biological fluids of patients with systemic inflammatory, autoimmune, or allergic disease, such as acute pancreatitis, rheumatoid arthritis, bronchial asthma, and allergic rhinitis. PLA2G1B has been thought to play major role in digestion of glycerophospholipids in nutrients, given its abundance in digestive organs (4). Since its expression has been observed in non-digestive organs including the lung, spleen, kidney, ovary, retina, brain, and neurons, its function may not limited to digestive role (5, 6). rhPLA2G1B has been maturated by cleaving the pro-peptide with trypsin, and the mature active form was further purified with ion-exchange column chromatograpy.

1. Webb, N. R. (2005) Cur. Opin. Lipid. 16:341.
2. Triggiani, M. et al. (2005) J. Allergy Clin. Immunol. 116:1000.
3. Murakami, M. and Kudo, I. (2004) Biol. Pharm. Bull. 27:1158.
4. de Haas, G. H. et al. (1968) Biochime. Biophysic. Acta. 159:118.
5. Kolko, M. et al. (2005) Cell. Mol. Neurobiol. 25:1107.
6. Kolko, M. et al. (2007) Acta Ophthalmol. Scand. 85:317.

Bioinformatics

• Gene Symbol: PLA2G1B
• Uniprot:
  • NP_000919()