Recombinant Human PON1 His-tag Fc Chimera Protein, CF Summary
with an N-terminal 6-His tag and a C-terminal Fc 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, CaCl2, 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, 5 mM CaCl2, pH 8.0
- Deionized Water
- Recombinant Human PON1 His-tag Fc Chimera (rhPON1) (Catalog 10175-PO)
- Substrate: Phenyl Acetate (7.88 M) (Sigma, Catalog # 108723)
- UV Plate (Catalog # 3635)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhPON1 to 10 µg/mL in Assay Buffer.
- Dilute Phenyl Acetate to 40 mM in deionized water.
- Load 50 µL of 10 µg/mL rhPON1 in a plate, and start the reaction by adding 50 µL of Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate.
- Read plate at a wavelength of 270 nm (absorbance) in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol|
|ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Using the extinction coefficient 1310 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD
- rhPON1: 0.5 µg
- Phenyl Acetate: 20 mM
Recombinant Human PON-1 His-tag Fc Chimera (Catalog # 10175-PO) ismeasured by its ability to hydrolyze phenyl acetate. The activity (orange) isover 3-fold higher than the competitor's PON-1 (green).
2 μg/lane of Recombinant Human PON1 His-tag Fc Chimera was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® blue staining, showing a band at ~74 kDa under reducing conditions.
Serum paraoxonase 1 (PON-1) is a member of the paraoxonase family which has three members: PON1, PON2, PON3. PON-1 is a lactonase (1,2) but has significant promiscuity (2,3) that allows hydrolysis of a variety of substrates including organophosphate triesters (including many pesticides), arylesters, cyclic carbamates, glucuronides and consequently also pharmaceuticals such as statins. PON-1 is a calcium-dependent, secreted, 43 kDa protein with a 6-bladed propeller structure and an active site gorge containing a His-His catalytic dyad (4). It is a homodimer and retains its hydrophobic signal sequence in the N-terminal region which enables its association with HDL, resulting in its localization predominantly in the plasma (5,6). Low serum PON1 and dysfunctional HDL is associated with many diseases with an inflammatory component including diabetes mellitus (7,8), rheumatoid arthritis, hepatic and renal diseases, psoriasis, and macular degeneration (9,10). PON1 shows a variety of atheroprotective properties (6,11,12) by metabolizing inflammatory lipid peroxides (13). PON-1 activity is low in coronary heart disease patients (14) and contributes to an increased risk of a major adverse cardiovascular event (15). PON-1 also protects against toxicity of organophosphorus compounds used as pesticides (16,17) thought to increase risk of neurodegenerative disorders such as Parkinson's (18) and Amyotrophic Lateral Sclerosis (19).
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