Western blot analysis of extracts from various cell lines using DHFR Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western blot analysis of extracts from 293T cells, either wild-type or DHFR knockdown, using DHFR Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western blot analysis of extracts from 293T cells, mock-transfected (-), transfected with DHFR-mycDDK (+) or transfected with DHFRL1/DHFR2-mycDDK (+) using DHFR Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
|REACTIVITY||H M R Mk|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
DHFR Antibody recognizes endogenous levels of total DHFR protein. This antibody does not cross-react with DHFRL1 protein.Species Reactivity:
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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