Recombinant Mouse Carboxylesterase 1/CES1 Protein, CF Summary
Details of Functionality |
Measured by its ability to hydrolyze p-nitrophenylacetate. The specific activity is >330 pmol/min/μg, as measured under the described conditions. |
Source |
Mouse myeloma cell line, NS0-derived mouse Carboxylesterase 1/CES1 protein His19-Leu565, with a C-terminal 6-His tag |
Accession # |
|
N-terminal Sequence |
His19 |
Protein/Peptide Type |
Recombinant Enzymes |
Gene |
Ces1g |
Purity |
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane |
Endotoxin Note |
<0.01 EU per 1 μg of the protein by the LAL method. |
Applications/Dilutions
Dilutions |
|
Theoretical MW |
62 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 |
55-60 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 Tris and NaCl. |
Purity |
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane |
Assay Procedure |
- Assay Buffer: 50 mM Tris, 150 mM NaCl, pH 7.5
- Reading Buffer: 50 mM Tris, pH 8.0
- Recombinant Mouse Carboxylesterase 1/CES1 (rmCES1) (Catalog # 7929-CE)
- Substrate: 4-Nitrophenyl acetate (4-NPA) (Sigma, Catalog # N-8130), 100 mM stock in Acetone
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rmCES1 to 12 ng/µL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- Load 50 µL of 12 ng/µL rmCES1 into a clear microplate in triplicate and start the reaction by adding 50 µL of 2 mM 4-NPA. Include a Substrate Blank consisting of 50 µL of Assay Buffer and 50 µL of 2 mM 4‑NPA.
- Incubate for 10 minutes at room temperature.
- Add 100 µL Reading Buffer to each well (this will not stop the reaction).
- Read immediately at a wavelength of 410 nm (bottom read) in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = |
Adjusted Abs* (OD) x Conversion Factor** (pmol/OD) |
Incubation time (min) x amount of enzyme (µg) |
*Adjusted for Substrate Blank **Derived using calibration standard 4-Nitrophenol (Sigma, Catalog # 241326). Per Well:
- rmCES1: 0.6 µg
- Substrate: 0.5 mM
|
Notes
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Mouse Carboxylesterase 1/CES1 Protein, CF
Background
Carboxylesterase 1 is a member of a large family of carboxylesterases that are responsible for the hydrolysis of ester and amide bonds (1, 2). They have broad substrate specificity ranging from small molecule esters such as phenylester to long chain fatty acid esters and thioesters. They play a major role as determinants of pharmacokinetic behavior for most therapeutic agents containing an ester. By de‑esterification, they can activate or inactivate the agents. They also participate in detoxification of drugs such as cocaine and heroin in serum and liver. The resulting de‑esterified metabolites are secreted out in urine. They can also detoxify organophosphate and carbamate analogues used in agrochemicals or chemical nerve agents, such as malathion, sarin, tabun, and VX. In addition to the hydrolytic activity, they can perform transesterification, a reaction important for cholesterol homeostasis. Carboxylesterase deficiency may be associated with non‑Hodgkin lymphoma or B‑cell lymphocytic leukemia. CES‑1 shares the serine hydrolase fold observed in other esterases (3). Ces1G is a rat and mouse specific protein that is expressed predominantly in liver, but also in kidney and lung (4).
-
Redinbo, M.R. and Potter, P.M. (2005) Drug Discovery Today 10:313.
-
Satoh, T. and Hosokawa, M. (2006) Chem. Biol. Interactions 162:195.
-
Fleming, C.D. et al. (2007) Biochemistry 46:5603.
-
Ellinghaus P. et al. (1998) Biochim. Biophys. Acta. 1397:175.
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