Recombinant Human Aldo-keto Reductase 1B10/AKR1B10, CF Summary
Ala2-Tyr316, with an N-terminal Met and 6-His 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, 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 Sodium Acetate, 0.1 M NaCl, pH 5.0
- Recombinant Human Aldo‑keto Reductase 1B10/AKR1B10 (rhAKR1B10) (Catalog # 7529-DH)
- beta -Nicotinamide adenine dinucleotide phosphate reduced, tetrasodium salt ( beta -NADPH) (Sigma, Catalog # N7505), 10 mM in deionized water
- 4-nitro-benzaldehyde (Fluka, Catalog # 72800), 200 mM in DMSO
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhAKR1B10 to 4 ng/μL in Assay Buffer.
- Prepare a Reaction Mixture containing 2 mM 4-nitro-benzaldehyde and 400 μM beta -NADPH in Assay Buffer.
- In a plate, load 50 μL of 4 ng/μL rhAKR1B10, and start the reaction by adding 50 μL of Reaction Mixture.
- Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Reaction 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 -1 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:
- rhAKR1B10: 0.2 μg
- 4-nitro-benzaldehyde: 1 mM
- beta -NADPH: 200 μM
Background: Aldo-keto Reductase 1B10/AKR1B10
Aldo-keto reductase family 1 member B10 (AKR1B10), also known as ARL-1 or aldose reductase related protein, is a cytosolic, NADPH-dependent oxidoreductase that reduces variety of aromatic and aliphatic aldehydes, dicarbonyl compounds, and some drug ketones (1). It is related to AKR1B1 in amino acid sequence and tertiary structure. However, its substrate specificity differs from AKR1B1 in its inability to reduce sugars and prostaglandin H2, and in its high catalytic efficiency for retinals, isoprenyl aldehydes, and cytotoxic aldehydes (2). It is primarily observed in the human colon, small intestine, and adrenal gland, with a low level in liver. Its up‑regulated expression is detected in hepatocellular carcinoma, cervical cancer, lung squamous cell carcinoma, and lung adenocarcinoma in smokers (3). Thus, it is considered as a potential diagnostic and/or prognostic marker in carcinomas and serum. The functional studies of this enzyme suggest that the up‑regulation of AKR1B10 is related to cell survival by reducing chemo-agents or metabolites in cells to less toxic reduced forms (4). Its inhibition may be a potential target for reducing the progression of cancers (5).
- Endo, S. et al. (2009) Arch. Biochem. Biophys. 487:1.
- Gallego, O. et al. (2007) Proc. Natl. Acad. Sci. USA 104:20764.
- Matsunaga, T. et al. (2012) Front. Pharmacol. 3:1.
- Wang, C. et al. (2009) J. Biol. Chem. 284:26742.
- Matsunaga, T. et al. (2011) Anticancer Drugs 22:402.
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