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- Table of Contents
MTHFD1 / C-1-tetrahydrofolate synthase, cytoplasmic · Western blot design guide
Design a Western Blot for MTHFD1
Source-linked MTHFD1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MTHFD1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use
MTHFD1 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~101.5 kDa | |
| Observed band | ~102 kDa | |
| Gel | 5–20% (catalog A02025-1) | |
| Positive control | Liver (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked MTHFD1 Western Blot Protocol Options
The A02025-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human 293T, rat brain, mouse brain, mouse kidney (catalog A02025-1) |
| Gel % | 5–20% (catalog A02025-1) |
| Load | 30 ug; reducing conditions (catalog A02025-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02025-1) |
| Membrane | nitrocellulose membrane (catalog A02025-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02025-1) |
| Primary antibody | A02025-1 · 0.25 μg/mL (catalog A02025-1) |
| Primary incubation | overnight at 4°C (catalog A02025-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02025-1) |
| Secondary incubation | 1.5 hour at RT (catalog A02025-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02025-1) |
| Detection | ECL (catalog A02025-1) |
Section 2
What Is the Expected MTHFD1 Western Blot Band Size?
MTHFD1 has a predicted mass of 101.5 kDa and an observed band near 102 kDa; the small difference has no established cause.
| Single band near 102 kDa | Matches the observed MTHFD1 band and its 101.5 kDa predicted monomer mass |
| Band near 203 kDa | Could reflect a homodimer if it survives sample preparation; confirm its identity |
| No band near 203 kDa | Consistent with dissociation of the UniProt annotated homodimer during SDS-PAGE |
| Several closely spaced bands near 102 kDa | Could reflect different phosphorylation states at annotated serines, but this migration pattern is unproven |
💡Expected MTHFD1 appearanceMTHFD1 has a predicted monomer mass of 101.5 kDa and an empirical band near 102 kDa; confirm band identity with an appropriate positive control or MTHFD1 depletion.
| UniProt predicted monomer mass | Places the expected monomer near 101.5 kDa |
| 935-residue sequence | Underlies the 101.5 kDa predicted monomer mass |
| Homodimer formation | Could yield a band near twice the monomer mass if the dimer persists during electrophoresis |
| Homodimer dissociation | Leaves the monomer near 102 kDa under denaturing conditions |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | MTHFD1 is cytoplasmic, so poor recovery or detection may account for its absence | Check lysate quality, loading and transfer with a positive control |
| Band higher than expected | A homodimer may persist if sample denaturation is incomplete | Compare fully denatured samples and confirm identity by MTHFD1 depletion |
| Band lower than expected | Proteolysis may have shortened the protein | Prepare fresh lysate with protease inhibitors and check the band with MTHFD1 depletion |
| Multiple bands | Phosphorylation is annotated, but distinct migrating forms are unproven | Use phosphatase treatment and MTHFD1 depletion to identify the bands |
| Weak or no signal | Insufficient cytoplasmic protein recovery or transfer may limit detection | Check lysate loading, transfer and a positive control |
| Fragments below expected size | Protein degradation may generate smaller immunoreactive products | Use fresh lysate with protease inhibitors and confirm identity by MTHFD1 depletion |
Experiment evidence
Western blot images showing MTHFD1 band
A02025-1 · MTHFD1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MTHFD1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MTHFD1 in Western blot, you can use liver lysate, which has high HPA expression.
Positive control: Liver (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: No tissue is listed as not detected, so use siRNA knockdown or a KO line for a negative control.
HPA tissue expression evidence for MTHFD1
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Higher expression tissues · candidate positive controls from IHC
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Liver | hepatocytes | High | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Cerebellum | cells in granular layer | Low | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Low | Protein (IHC) | HPA → |
| Soft tissue | chondrocytes | Low | Protein (IHC) | HPA → |
Section 3
Advanced MTHFD1 Western Blot Tips
Deeper troubleshooting and optimisation questions for MTHFD1, answered from its protein features.
How should MTHFD1 band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
Could MTHFD1 isoforms explain multiple bands?
Isoforms · Only one isoform is supplied, with no alternative sequence. These features do not support assigning additional bands to MTHFD1 isoforms.
Which MTHFD1 modifications matter when interpreting bands?
PTM · UniProt lists N-acetylmethionine at position 1 and phosphoserine at positions 318, 413, and 490. These are UniProt sequence coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible band shift.
Does this guide establish induction of MTHFD1?
Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.
What transfer method to use for MTHFD1 Western blot?
Transfer · Choose conditions that transfer a protein near 102 kDa, and verify transfer in that region by staining the membrane and checking the gel afterward. The supplied features do not specify a preferred transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02025-1 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.
How should total MTHFD1 be quantified?
Quantitation · Measure the approximately 102 kDa band in the assay's linear range and normalize to total lane protein. MTHFD1 is listed as cytoplasmic. Total-band intensity does not distinguish phosphorylation at UniProt Ser318, Ser413, or Ser490.
Should MTHFD1 migrate near its predicted mass?
Interpretation · The predicted mass is 101.5 kDa, and the reported band is approximately 102 kDa. The listed modifications do not establish a visible shift or explain a mass difference.
Compare them with the expected approximately 102 kDa band. One isoform is supplied, and no signal peptide, propeptide, or glycosylation sites are listed. The listed modifications alone cannot identify an unexpected band; verify its identity experimentally.
UniProt describes MTHFD1 as a homodimer. That feature alone does not identify a higher Western-blot band as a dimer. Compare the band with the approximately 102 kDa monomer and verify its identity experimentally.
Compare them with the expected approximately 102 kDa band. One isoform is supplied, and no signal peptide, propeptide, or glycosylation sites are listed. The listed modifications alone cannot identify an unexpected band; verify its identity experimentally.
UniProt describes MTHFD1 as a homodimer. That feature alone does not identify a higher Western-blot band as a dimer. Compare the band with the approximately 102 kDa monomer and verify its identity experimentally.
Boster reagents
MTHFD1 Western Blot Antibodies
Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.
Real WB data
Anti-MTHFD1 Antibody Picoband®
Cat # A02025-1
The catalog reports one anti-MTHFD1 antibody, A02025-1, with reported human, mouse, and rat reactivity. Its Western blot image reports a band at approximately 102 kDa, matching the expected size. The supplied evidence is limited to the product blot.
Which to pick: A02025-1 is the only listed option. Its Western blot used HeLa and 293T cells, rat brain, and mouse brain and kidney lysates at 0.25 μg/mL. These examples document the tested samples, not every tissue or condition.
Source: BosterBio MTHFD1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P11586.
- Human Protein Atlas. MTHFD1 tissue expression.
- PMC6259576 — target-verified WB comparison
- PMC10132981 — target-verified WB comparison
- PMC6952269 — target-verified WB comparison