| DHFR, also known as Dihydrofolate reductase, is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as electron donor, which can be converted to the kinds of tetrahydrofolate cofactors used in 1-carbon transfer chemistry. Dihydrofolate reductase deficiency has been linked to megaloblastic anemia. Recombinant Dihydrofolate reductase protein, fused to His-tag at N-terminus, , expressed in E. coli and purified by using conventional chromatography techniques. Recombinant protein corresponding to aa1-187 from human DHFR, fused to His-Tag at N-terminal, expressed in E. coli. Amino Acid Sequence: MGSSHHHHHH SSGLVPRGSH MVGSLNCIVA VSQNMGIGKN GDLPWPPLRN EFRYFQRMTT TSSVEGKQNL VIMGKKTWFS IPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSS VYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKF EVYEKND Biological Activity: Specific activity is 1.5-2.5 units/ml and was obtained by measuring the oxidation of NADPH in absorbance at 340 nm during reaction. One unit will convert 1.0 umole of 7,8 dihydrofolate and beta-NADPH to 5,6,7,8-tetrahydrofolate and beta-NADP per minute at pH 6.5 at 25C. Activity Assay: 1. Prepare a 3.2ml reaction mixture into a suitable container: The final concentrations are 50mM potassium phosphate, 0.72mM DHFA, 0.1mM beta-NADPH, 0.003% (w/v) BSA. 2. Equilibrate to 25C and monitor the A340nm until the value is constant using a spectrophotometer. 3. Add 2.5ug of recombinant DHFR into reaction mixture and mix immediately. 4. Record the increase in A340nm for 2 minutes. Applications: Suitable for use in ELISA and Western Blot. Other applications not tested. Recommended Dilution: Optimal dilutions to be determined by the researcher. Storage and Stability: Aliquot to avoid repeated freezing and thawing and freeze at -70C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Aliquots are stable for at least 6 months. |
