|REACTIVITY||H M R Mk|
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human DHFR protein. Antibodies are purified by protein A and peptide affinity chromatography.
Dihydrofolate reductase (DHFR) catalyzes tetrahydrofolate regeneration through the reduction of dihydrofolate using NADPH as a cofactor (1). As a key enzyme in folate metabolism, DHFR is ubiquitously expressed in the mitochondria, and is essential for the synthesis of purines, pyrimidines, and some amino acids (2). DHFR is capable of translational autoregulation by binding within the coding region of its own mRNA sequence to repress cellular DHFR protein levels (3). Mutations in the DHFR gene are known to cause inborn errors of folate metabolism resulting in megaloblastic anemia, pancytopenia, and severe cerebral folate deficiency (4). Because tetrahydrofolate is essential for DNA synthesis, cell growth, and proliferation, DHFR is a target of chemotherapeutic agents (e.g., methotrexate and pemetrexed) used in the treatment of many cancer types (5). Increased expression of DHFR has also been identified as a potential mechanism for tumor resistance to methotrexate, and therefore has been utilized as a clinical biomarker to predict patient responsiveness to folate antagonists (6,7).
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