Aldose reductase (AKR1B1) is a member of the aldo-keto reductase (AKR) superfamily and catalyzes the NADPH-dependent reduction of a wide variety of carbonyl-containing compounds to their corresponding alcohols. This protein is implicated in the development of diabetic complications by catalyzing the reduction of glucose to sorbitol.
Human Aldose reductase (AKR1B1) protein was expressed in E. coli and purified by using conventional chromatography techniques.
0.5-0.6 units/mg protein. Enzymatic activity was confirmed by measuring the amount of enzyme catalyzing the oxidation of 1 micromole NADPH per minute at 25 degrees C.
1. Prepare a 750ul reaction mix into a suitable container. The final concentrations of the reaction mix components should be: 0.1M sodium phosphate (pH7.0) and 0.3mM NADPH.
2. Add 50ul of recombinant AKR1B1 solution with various concentrations (2.5ug, 5ug, 10ug) in 750ul reaction buffer.
3. Mix by inversion and Incubate at 25 degrees C for 2.5 minutes.
4. Add 200ul of 50 mM DL-glyceraldehyde substrate and immediately mix by inversion.
5. Record the increase in A340nm for 3 minutes.
MASRLLLNNG AKMPILGLGT WKSPPGQVTE AVKVAIDVGY RHIDCAHVYQ NENEVGVAIQ EKLREQVVKR EELFIVSKLW CTYHEKGLVK GACQKTLSDL KLDYLDLYLI HWPTGFKPGK EFFPLDESGN VVPSDTNILD TWAAMEELVD EGLVKAIGIS NFNHLQVEMI LNKPGLKYKP
AVNQIECHPY LTQEKLIQYC QSKGIVVTAY SPLGSPDRPW AKPEDPSLLE DPRIKAIAAK HNKTTAQVLI RFPMQRNLVV IPKSVTPERI AENFKVFDFE LSSQDMTTLL SYNRNWRVCA LLSCTSHKDY PFHEEF
(same/less than)1.0 EU per 1 ug of protein (LAL)
Storage and Stability:
For long-term storage, aliquot and store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.