Recombinant Human Aldo-keto Reductase 1C1/AKR1C1 Protein, CF Summary
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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, NaCl, DTT 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, 500 mM NaCl, pH 8.5
- Substrate Buffer: 50 mM Tris, 500 mM NaCl, 20% (v/v) ethanol, pH 8.5
- Recombinant Human Aldo-keto Reductase 1C1/AKR1C1 (rhAKR1C1) (Catalog # 6529-DH)
- S-1-Indanol (Sigma, Catalog # 323128), 200 mM in DMSO
- beta -Nicotinamide adenine dinucleotide phosphate (NADP+) (Sigma, Catalog # N5755), 50 mM in deionized water
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhAKR1C1 to 40 ng/μL in Assay Buffer.
- Dilute Indanol to 4 mM in Substrate Buffer.
- Dilute NADP+ to 2 mM in Substrate Buffer.
- Combine equal volumes of 4 mM Indanol and 2 mM NADP+ to form the Substrate Mixture.
- Load 50 μL of 40 ng/μL rhAKR1C1 into the microplate and start the reaction by adding 50 μL of Substrate Mixture. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
- Read at an absorbance of 340 nm 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 6270 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD. Per Well:
- rhAKR1C1: 2 μg
- Indanol: 1 mM
- NADP+: 0.5 mM
Background: Aldo-keto Reductase 1C1/AKR1C1
AKR1C1 (20-alpha -hydroxysteroid dehydrogenase, 20‑ alpha ‑HSD) is a member of aldo-keto reductase (AKR) superfamily. AKRs perform the NAD(P)H-dependent reduction of carbonyl groups (1). Four AKR1C isoforms (AKR1C1-C4) are known to exist in humans. They are all highly expressed in the liver. Three isoforms, excluding AKR1C4, have a wider expression pattern including prostate, testes, uterus, mammary gland, and haemopoietic progenitors (2). These enzymes are able to accept various natural steroids as substrates, including 3-, 7-, and 20‑ketosteroids (3). They can also activate prodrugs such as synthetic steroid hormone tibolone by converting it into active 3 alpha / beta -hydroxy form (4). They are recognized as phase I drug-metabolizing enzymes involved in the maintenance of steroid homeostasis, prostaglandin metabolism, and metabolic activation of polycyclic aromatic hydrocarbons (5). Their reactions introduce a hydroxyl group into the product making it available for sulfonation and glucuronidation by phase II enzyme. Elevated expression of these enzymes is related to cancer with hormone-dependent malignancies (6, 7). Increased levels of expression of AKR1C1 parallels increased cell proliferation activity in human colon cancer cells. It has been shown to be associated with oncogenic potential and proproliferative effects. It is also involved in cancer cell chemoresistance.
- Jez, J.M. et al. (1997) Biochem J. 326:499.
- Penning, T.M. et al. (2000) Biochem. J. 351:67.
- Rizner, T.L. et al. (2003) Endocrinology. 144:2922.
- Steckelbroeck, S. et al. (2006) J. Pharmacol. Exp. Ther. 316:1300.
- Penning, T.M. et al. (2004) Mol. Cell. Endocrinol. 1784:1342.
- Penning, T.M. and M.C. Byrns (2009) Ann. N. Y. Acad. Sci. 1155:33.
- Baumann, D.R. et al. (2004) Drug News Perspect. 17:563.
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