MTHFR Antibody (Rabbit mAb) [N5P2]

Application: Reactivity: Mouse, Human

Usage Information

Dilution
WB1:1000 IP1:30 IF1:100

Application
WB, IP, IF

Reactivity
Mouse, Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
75 kDa 42,75 kDa,90-120 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Human thyroid carcinoma tissue; Human kidney tissue; Human fetal brain tissue; Human fetal heart tissue; Human fetal kidney tissue; Human spleen tissue; Human fetal liver tissue; Mouse placenta tissue; Mouse kidney tissue; Jurkat cells; RAW 264.7 cells; HT-29 cells; HEK-293 cells; NIH/3T3 cells
Negative Control

Datasheet & SDS

Biological Description

Specificity
MTHFR Antibody (Rabbit mAb) [N5P2] detects endogenous levels of total MTHFR protein.

Clone
N5P2

Synonym(s)
Methylenetetrahydrofolate reductase (NADPH), MTHFR

Background
Methylenetetrahydrofolate reductase, MTHFR, is a flavoprotein that commits folate-derived one-carbon units to the methionine cycle by catalyzing the physiologically irreversible reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, non-covalently binding FAD as an essential cofactor and shuttling electrons from NADPH through a ping-pong bi-bi catalytic mechanism. The eukaryotic enzyme is homodimeric, with each monomer built from an N-terminal catalytic domain, adopting a conserved TIM-barrel fold that houses the FAD cofactor, connected through an extended interdomain linker to a structurally distinct C-terminal regulatory domain that mediates dimerization and confers allosteric feedback inhibition, a regulatory domain absent from bacterial MTHFR homologs. Cryo-electron microscopy structures of the full-length human enzyme reveal that MTHFR shifts between two conformational states depending on which methyl-cycle metabolite occupies its regulatory domain: in the active state, a single molecule of S-adenosylhomocysteine bound to the regulatory domain leaves the catalytic domain flexible and its active site exposed, whereas in the inhibited state the regulatory pocket is remodeled to expose a second, previously occluded binding site that accommodates two molecules of S-adenosylmethionine, and this dual-SAM-bound conformation rearranges the interdomain linker so that specific tyrosine residues insert into the active site and directly position over the FAD cofactor, physically blocking substrate access. This SAM-dependent allosteric inhibition is tunable through phosphorylation: multiple serine/threonine residues in the N-terminal region of MTHFR are phosphorylated by the kinases DYRK1A/2 and GSK3A/B, and phosphorylated MTHFR carries a lower inhibitory constant for SAM, making the phosphorylated enzyme more sensitive to allosteric shutdown at a given SAM concentration, while MTHFR phosphorylation itself is maintained by adequate cellular SAM levels sensed through the same C-terminal regulatory domain, forming a feedback loop linking one-carbon metabolic status to enzyme phosphorylation state. CRISPR knock-in cell lines engineered to abolish MTHFR phosphorylation show elevated basal production of 5-methyltetrahydrofolate and fail to further increase this output in response to homocysteine treatment, unlike parental cells, directly demonstrating that phosphorylation-dependent SAM sensitivity is required for MTHFR to dynamically adjust its output according to cellular homocysteine and methionine demand rather than operating at a fixed catalytic rate. Because SAM serves as the shared allosteric inhibitor of MTHFR and allosteric activator of cystathionine beta-synthase, this single small molecule reciprocally controls flux between the remethylation and transsulfuration branches of homocysteine metabolism. The common C677T polymorphism reduces enzyme stability and activity, and this reduced-function variant is associated with elevated homocysteine and increased risk of cardiovascular and neurological disease.

Background

Methylenetetrahydrofolate reductase, MTHFR, is a flavoprotein that commits folate-derived one-carbon units to the methionine cycle by catalyzing the physiologically irreversible reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, non-covalently binding FAD as an essential cofactor and shuttling electrons from NADPH through a ping-pong bi-bi catalytic mechanism. The eukaryotic enzyme is homodimeric, with each monomer built from an N-terminal catalytic domain, adopting a conserved TIM-barrel fold that houses the FAD cofactor, connected through an extended interdomain linker to a structurally distinct C-terminal regulatory domain that mediates dimerization and confers allosteric feedback inhibition, a regulatory domain absent from bacterial MTHFR homologs. Cryo-electron microscopy structures of the full-length human enzyme reveal that MTHFR shifts between two conformational states depending on which methyl-cycle metabolite occupies its regulatory domain: in the active state, a single molecule of S-adenosylhomocysteine bound to the regulatory domain leaves the catalytic domain flexible and its active site exposed, whereas in the inhibited state the regulatory pocket is remodeled to expose a second, previously occluded binding site that accommodates two molecules of S-adenosylmethionine, and this dual-SAM-bound conformation rearranges the interdomain linker so that specific tyrosine residues insert into the active site and directly position over the FAD cofactor, physically blocking substrate access. This SAM-dependent allosteric inhibition is tunable through phosphorylation: multiple serine/threonine residues in the N-terminal region of MTHFR are phosphorylated by the kinases DYRK1A/2 and GSK3A/B, and phosphorylated MTHFR carries a lower inhibitory constant for SAM, making the phosphorylated enzyme more sensitive to allosteric shutdown at a given SAM concentration, while MTHFR phosphorylation itself is maintained by adequate cellular SAM levels sensed through the same C-terminal regulatory domain, forming a feedback loop linking one-carbon metabolic status to enzyme phosphorylation state. CRISPR knock-in cell lines engineered to abolish MTHFR phosphorylation show elevated basal production of 5-methyltetrahydrofolate and fail to further increase this output in response to homocysteine treatment, unlike parental cells, directly demonstrating that phosphorylation-dependent SAM sensitivity is required for MTHFR to dynamically adjust its output according to cellular homocysteine and methionine demand rather than operating at a fixed catalytic rate. Because SAM serves as the shared allosteric inhibitor of MTHFR and allosteric activator of cystathionine beta-synthase, this single small molecule reciprocally controls flux between the remethylation and transsulfuration branches of homocysteine metabolism. The common C677T polymorphism reduces enzyme stability and activity, and this reduced-function variant is associated with elevated homocysteine and increased risk of cardiovascular and neurological disease.

References
https://pubmed.ncbi.nlm.nih.gov/30862944/ https://pubmed.ncbi.nlm.nih.gov/38622112/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%