Recombinant Human PLA2G1B Protein, CF Summary
Product Specifications
Ala23-Ser148, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
5018-PL
| Formulation | Supplied as a 0.2 μm filtered solution in Sodium Acetate and NaCl. |
| 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 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
- Thaw Substrate at 37 °C for five minutes. Mix well.
- Dilute rhPLA2G1B to 0.02 ng/µL in Assay Buffer.
- Dilute Substrate to 100 µM in Substrate Buffer.
- 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.
- 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.
- 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.
- rhPLA2G1B: 0.001 µg
- Substrate: 50 µM
Reconstitution Calculator
Background: PLA2G1B
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.
- Webb, N. R. (2005) Cur. Opin. Lipid. 16:341.
- Triggiani, M. et al. (2005) J. Allergy Clin. Immunol. 116:1000.
- Murakami, M. and Kudo, I. (2004) Biol. Pharm. Bull. 27:1158.
- de Haas, G. H. et al. (1968) Biochime. Biophysic. Acta. 159:118.
- Kolko, M. et al. (2005) Cell. Mol. Neurobiol. 25:1107.
- Kolko, M. et al. (2007) Acta Ophthalmol. Scand. 85:317.
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