Recombinant Human COX-2 His-tag Protein, CF Summary
Product Specifications
Ala18-Leu604
with a C-terminal 6-His tag
Analysis
Product Datasheets
10465-CX
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.
10465-CX
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, CHAPS 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 Procedure
- Assay Buffer A: 50 mM Tris, 5 µM Hemin (Sigma, Catalog # H9039), pH 8.0
- Assay Buffer B: 50 mM Tris, pH 8.0
- Recombinant Human COX-2 (rhCOX-2) (Catalog # 10465-CX)
- Substrate Component 1: Arachidonic acid (Sigma, Catalog # 10931), 10 mM stock in DMSO
- Substrate Component 2: Amplex Ultra Red (AUR) (Molecular Probes, Catalog # A36006), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhCOX-2 to 5 ng/µL in Assay Buffer A.
- Prepare Substrate Mixture 50 µM Arachidonic acid and 100 µM AUR in Assay Buffer B.
- Load into a plate 50 µL of diluted rhCOX-2. Also create a Substrate Blank by loading 50 µL of Assay Buffer A.
- Add 50 µL of Substrate Mixture to all wells and incubate at room temperature for 1 minute.
- Read at excitation and emission wavelengths of 540 nm and 585 nm (top read), respectively in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU) |
| Incubation time (min) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard Resorufin (Sigma, Catalog # R3257)
- rhCOX-2: 0.25 µg
- Arachidonic acid: 25 µM
- AUR: 50 µM
Scientific Data
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2 μg/lane of Recombinant Human COX-2 His-tag Protein (Catalog # 10465-CX) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 65-71 kDa.
Background: COX-2
Cyclooxygenase 2 (COX-2), also known as Prostaglandin synthase 2 (PTGS2), is a dimeric, heme-dependent enzyme where the dimer is formed from two monomers each containing an N-terminal EGF-like domain, a membrane binding domain and a C-terminal catalytic domain (1). COX-2 catalyzes the conversion of arachidonate to prostaglandin H2 through a two-step reaction, representing the rate-limiting enzyme in the biosynthesis of prostanoids. Altered levels of prostaglandins are associated with several disease pathologies. There are two major isozymes of PTGS with slightly differing substrate preferences; PTGS1 represents the constitutively expressed form while PTGS2 basal expression is present in a limited number of tissues (2) such as inflammatory cells. PTGS2 is upregulated in chronic and acute inflammation as well as in most types of cancers (3) resulting in increased rate of recurrence (4), reduction in survival (5), and increased resistance to chemo and radiotherapy (6) through the role it plays in multiple pathways and mechanisms (7). Inhibition of PTGS2 is a promising therapeutic target to reduce cancer risk (8, 9). COX-2 is a pharmacological target of nonsteroidal anti-inflammatory drugs (NSAIDS) and COX-2 selective inhibitors (coxibs) as well as other categories of inhibitors through multiple mechanisms of action (1). It remains an attractive target for novel specific inhibitor development as inhibition shows equivalent efficacy to use of conventional NSAIDs, but with reduced negative side effects (10, 11).
- Orlando, B.J. et al. (2015) J. Struct. Biol. 189:62.
- Su, C.W. et al. (2016) Crit. Rev. Oncol. Hematol. 107:33.
- Gurram, B. et al. (2018) Anal. Chem. 90:5187.
- Montezuma, M.A.P. et al. (2018) Pathol. Res. Pract. 214:907.
- Hoing, B. et al. (2018) Oncotarget 9:8415.
- Dong, X.F. et al. (2018) Clin. Cancer Res. 24:3204.
- Hashemi Goradel, N. et al. (2019) J. Cell Physiol. 234:5683.
- Brizzolara, A. et al. (2017) Cancer Letters 400:9.
- Shi, L. et al. (2018) ACS Appl. Mater. Interfaces 10:8555.
- Ferrer, M.D. et al. (2019) Curr. Med. Chem. 26:3225.
- Carullo, G. et al. (2016) Medchemcomm. 8:492.
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