Western blot analysis of extracts from various cell lines using MTHFD1L (D8T7L) 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 datasheet or 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 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 October 2016
Protocol Id: 10
MTHFD1L (D8T7L) Rabbit mAb recognizes endogenous levels of total MTHFD1L protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala264 of human MTHFD1L protein.
NADP+ dependent methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) is a mitochondrial enzyme that catalyzes the production of formate from 10-formyl-tetrahydrofolate, the last step in one-carbon (1-C) flow from mitochondria to cytoplasm (1,2). These one-carbon end products are required for de novo synthesis of thymidylate and purines. In the mitochondria, these essential one-carbon products are formed by a series of reactions catalyzed by a pair of enzymes (MTHFD2 and MTHFD1L), but by the trifunctional MTHFD1 enzyme in the cytoplasm (3). The 10-formyl-tetrahydrofolate synthetase MTHFD1L is widely expressed in most adult tissues and at all stages of mammalian embryonic development (1). Research studies using MTHFD1L knockout mice indicate that MTHFD1L plays an essential role in neural tube formation; mice lacking MTHFD1L displayed neural tube and craniofacial defects leading to embryonic lethality (4).
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