Recombinant Human

Aldo-keto Reductase 1B10/AKR1B10 Catalog Number: 7529-DH

DESCRIPTION Source Spodoptera frugiperda, Sf 21 (baculovirus)­derived Ala2­Tyr316, with an N­terminal Met and 6­His tag Accession # O60218

N­terminal Sequence Inconclusive result, Met predicted. Protein identity confirmed by MS analysis of tryptic fragments Analysis Predicted Molecular 37 kDa Mass

SPECIFICATIONS SDS­PAGE 37­38 kDa, reducing conditions

Activity Measured by the disappearance of NADPH during the reducation of 4­nitrobenzaldehyde. The specific activity is >1,000 pmol/min/μg, as measured under the described conditions.

Endotoxin Level <0.10 EU per 1 μg of the protein by the LAL method.

Purity >90%, by SDS­PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane. Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Glycerol. See Certificate of Analysis for details.

Activity Assay Protocol Materials l Assay Buffer: 50 mM Sodium Acetate, 0.1 M NaCl, pH 5.0 l Recombinant Human Aldo­keto Reductase 1B10/AKR1B10 (rhAKR1B10) (Catalog # 7529­DH) l β­Nicotinamide adenine dinucleotide phosphate reduced, tetrasodium salt (β­NADPH) (Sigma, Catalog # N7505), 10 mM in deionized water l 4­nitro­benzaldehyde (Fluka, Catalog # 72800), 200 mM in DMSO l 96­well Clear Plate (Costar, Catalog # 92592) l Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Assay 1. Dilute rhAKR1B10 to 4 ng/μL in Assay Buffer. 2. Prepare a Reaction Mixture containing 2 mM 4­nitro­benzaldehyde and 400 μMβ­NADPH in Assay Buffer. 3. In a plate, load 50 μL of 4 ng/μL rhAKR1B10, and start the reaction by adding 50 μL of Reaction Mixture. 4. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Reaction Mixture. 5. Read at an absorbance of 340 nm in kinetic mode for 5 minutes. 6. Calculate specific activity: Adjusted V * (OD/min) x ­1 x well volume (L) x 1012 pmol/mol Specific Activity (pmol/min/µg) = max ext. coeff** (M­1cm­1) x path corr.*** (cm) x amount of enzyme (µg)

*Adjusted for Substrate Blank **Using the extinction coefficient 6270 M­1cm­1 ***Using the path correction 0.32 cm Note: the output of many spectrophotometers is in mOD Final Assay Per Well: Conditions l rhAKR1B10: 0.2 μg l 4­nitro­benzaldehyde: 1 mM l β­NADPH: 200 μM

PREPARATION AND STORAGE 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 freeze­thaw cycles. l 6 months from date of receipt, ­20 to ­70 °C as supplied. l 3 months, ­20 to ­70 °C under sterile conditions after opening.

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Recombinant Human

Aldo-keto Reductase 1B10/AKR1B10 Catalog Number: 7529-DH

BACKGROUND Aldo­keto reductase family 1 member B10 (AKR1B10), also known as ARL­1 or aldose reductase related protein, is a cytosolic, NADPH­dependent 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 up­regulated 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 up­regulation of AKR1B10 is related to cell survival by reducing chemo­agents or metabolites in cells to less toxic reduced forms (4). Its inhibition may be a potential target for reducing the progression of cancers (5).

References: 1. Endo, S. et al. (2009) Arch. Biochem. Biophys. 487:1. 2. Gallego, O. et al. (2007) Proc. Natl. Acad. Sci. USA 104:20764. 3. Matsunaga, T. et al. (2012) Front. Pharmacol. 3:1. 4. Wang, C. et al. (2009) J. Biol. Chem. 284:26742. 5. Matsunaga, T. et al. (2011) Anticancer Drugs 22:402.

PRODUCT SPECIFIC NOTICES Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

Rev. 2/6/2018 Page 2 of 2