Western blot analysis of extracts from Daudi, Raji, and MOLT-4 cells using MTHFR (D1E4V) Rabbit mAb.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: 10
MTHFR (D1E4V) Rabbit mAb recognizes endogenous levels of total MTHFR protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro39 of human MTHFR protein.
Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in one-carbon metabolism, catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-methyltetrahydrofolate donates its methyl group for remethylation of homocysteine to methionine. Methionine is further converted to S-adenosylmethionine (SAM), a major reactive methyl carrier. DNA methyltransferases and histone methyltransferases use SAM to methylate DNA and histones with concomitant conversion of SAM to S-adenosylhomocysteine (SAH) (1, 2). In addition, MTHFR is inhibited by SAM and this feedback inhibition is partially reduced by SAH (3). Metabolically regulated levels of SAM and SAM/SAH ratio are shown to predict histone methylation levels, indicating the important role of enzymes in one-carbon metabolism including MTHFR in determining histone methylation status (4).
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