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FOXC1 · Western blot design guide
Design a Western Blot for FOXC1
Real validated FOXC1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-FOXC1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
FOXC1 Western Blot Experimental Design Guide
Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~56.8 kDa | |
| Observed band | Approximately 75 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubiquitinated | |
| Caveat | Phosphorylation-induced mobility shift | |
| Regulation | Estrogen up | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated FOXC1 Western Blot Protocols
Literature-validated Western blot parameters for FOXC1 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human 293T . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-FOXC1 antigen affinity purified polyclonal antibody (A00962-2) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for FOXC1 at approximately 75 kDa. The expected band size for FOXC1 is at 53 kDa |
| Gel % | 10–12% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 75 kDa |
Section 2
What Is the Expected FOXC1 Western Blot Band Size?
FOXC1 has a 56.8 kDa predicted backbone but runs at approximately 75 kDa on Western blot, mainly due to phosphorylation and Ubl conjugation, not glycosylation or dimerization.
| Single band at approximately 75 kDa in nuclear or whole-cell lysate | This is the expected native FOXC1 band; it runs well above the 56.8 kDa predicted mass due to post-translational modification rather than a distinct species |
| No band near twice the predicted mass | FOXC1 functions as a monomer with no annotated inter-chain disulfide bonds, so a dimer-sized band is not expected |
| Single band, no doublet or ladder of isoform-sized bands | Only one FOXC1 isoform is annotated, so multiple isoform-driven bands are not expected |
| No smaller fragment well below the ~57 kDa predicted backbone | FOXC1 has no signal peptide or propeptide, so there is no smaller cleaved/mature product to look for |
| Sharp, well-resolved band rather than a smear | FOXC1 has no annotated glycosylation sites, so the band should stay sharp rather than smearing like a heterogeneously glycosylated protein |
| Band detected in nuclear-enriched or whole-cell lysate, not in culture supernatant | FOXC1 is a nuclear transcription factor, not a secreted protein, so signal is expected in cell lysates rather than conditioned media |
💡Expected FOXC1 appearanceExpect a single sharp FOXC1 band at approximately 75 kDa in nuclear or whole-cell lysate, well above the 56.8 kDa predicted mass due to phosphorylation and Ubl conjugation, not glycosylation, cleavage, or dimerization.
| Predicted mass (UniProt, 56.8 kDa / 553 aa) | Sets the baseline unmodified-backbone expectation that the observed native band runs above |
| Phosphorylation (5 annotated phosphoserine sites, e.g. Ser235, Ser241, Ser272) | Marks FOXC1 as a phosphoprotein; phosphorylated species commonly migrate more slowly than the unmodified backbone, shifting the apparent band upward |
| Ubl (ubiquitin-like) conjugation | Covalent attachment of a ubiquitin-like modifier adds mass to a portion of the protein pool, pushing its apparent size above the predicted 56.8 kDa |
| Monomer with no annotated disulfide bonds | No inter-chain disulfide-linked dimer forms, so the band should not double under non-reducing conditions |
| Single annotated isoform | No alternate splice forms exist to generate additional bands at different sizes |
| No signal peptide or propeptide | No proteolytic maturation step occurs, so no smaller cleaved fragment is expected relative to full-length FOXC1 |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | FOXC1 is nuclear-localized, so standard whole-cell lysis that under-solubilizes the nuclear compartment can leave it under-extracted | Use a lysis protocol with adequate nuclear extraction (e.g., RIPA with sonication or a nuclear extraction kit) and load a nuclear-enriched fraction |
| Band higher than expected | Phosphorylation and Ubl conjugation shift FOXC1's apparent mass above the 56.8 kDa predicted backbone, producing the ~75 kDa band seen on QC blots | Treat the ~75 kDa band as the expected native signal rather than comparing directly to the calculated 56.8 kDa mass |
| Band lower than expected | Bacterially expressed recombinant FOXC1 lacks phosphorylation and Ubl conjugation, so it runs closer to the 56.8 kDa predicted mass | Compare lysate samples to a mammalian-expressed or native positive control rather than a bacterial recombinant standard |
| Multiple bands | Partial phosphorylation or Ubl conjugation across the FOXC1 pool can produce closely spaced bands instead of one uniform species | Treat lysate with phosphatase to collapse modification-driven bands and confirm the single predicted backbone species |
| Weak or no signal | FOXC1 is a lower-abundance nuclear transcription factor, so low-expressing cell lines or insufficient nuclear enrichment give weak signal | Increase protein loading, use a nuclear extraction protocol, and confirm expression in a known FOXC1-positive line |
| Fragments below expected size | FOXC1 has no annotated propeptide or signal peptide, so smaller fragments are not a normal maturation product and instead suggest degradation | Add protease inhibitors during lysis and keep samples cold throughout preparation to prevent degradation artifacts |
Experiment evidence
Western blot images showing FOXC1 band
A00962-2 · FOXC1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for FOXC1 Western blot
🧪For positive controls for FOXC1 in Western blot, you can use a FOXC1-overexpressing cell lysate (e.g., HEK293 or HeLa cells transiently transfected with a FOXC1 expression construct), since no Human Protein Atlas expression data is available for this gene.
Positive control: FOXC1-transfected HEK293 cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin antibodies alongside a total-protein stain (stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: As a nuclear transcription factor with no HPA expression profile provided, a validated positive/negative tissue pair cannot be identified, so an overexpression lysate plus siRNA/KO validation is the more feasible route to confirm antibody specificity.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced FOXC1 Western Blot Tips
Deeper troubleshooting and optimisation questions for FOXC1, answered from its protein features.
Why does FOXC1 run at 75 kDa, not 56.8 kDa?
FOXC1 is a phosphoprotein with 5 modified residues plus ubiquitin-like conjugation reported in its UniProt entry. Added phosphate groups and SUMO/ubiquitin-like moieties reduce SDS binding and slow migration on standard Tris-glycine gels, so the observed ~75 kDa band is consistent with the unmodified 56.8 kDa core protein carrying these modifications rather than a nonspecific band.
Should I expect multiple FOXC1 isoform bands?
No. UniProt lists only one FOXC1 isoform with no alternative splice variants annotated, so a single specific band (allowing for the PTM-driven shift to ~75 kDa) is expected. Additional lower-molecular-weight bands are more likely degradation products or nonspecific signal than genuine isoforms.
Could FOXC1 phosphorylation affect signal or migration?
Yes. FOXC1 carries 5 annotated modified residues and is keyword-tagged Phosphoprotein and Ubl conjugation. These modifications can shift apparent molecular weight and, if the epitope overlaps a modified residue, reduce or alter antibody binding. If bands look faint or diffuse, consider a phosphatase treatment control to confirm the shift is PTM-driven.
What blocking buffer suits FOXC1 detection?
Use 5% BSA in TBST rather than milk, since milk casein can interfere with detection of phosphorylated epitopes on this phosphoprotein. FOXC1 is a low-abundance nuclear transcription factor, so extended blocking (1 hour) and thorough washing help minimize background against the specific ~75 kDa signal.
What transfer method to use for FOXC1 Western blot?
Standard wet or semi-dry transfer to PVDF works well, since FOXC1's predicted mass (56.8 kDa) and observed band (~75 kDa) both transfer efficiently at typical conditions. No extended transfer time is needed, as FOXC1 is not a large or membrane-embedded protein; it lacks a signal peptide and is nuclear-localized.
What loading control fits FOXC1 quantitation?
Because FOXC1 localizes to the nucleus, use a nuclear loading control such as Lamin B1 or Histone H3 rather than a cytoplasmic marker like GAPDH, especially if lysates are fractionated. This ensures normalization reflects nuclear protein input accurately for this transcription factor.
What explains extra bands beyond the main FOXC1 signal?
FOXC1 functions as a monomer but interacts with partners like GLI2 and C1QBP; extra bands are unlikely to reflect stable complexes surviving SDS-PAGE. More likely explanations are partially modified forms (phosphorylated or ubl-conjugated species running near 75 kDa) or degradation products below the predicted 56.8 kDa mass.
Boster reagents
Best FOXC1 Western Blot Antibodies
BosterBio's FOXC1 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-FOXC1 Antibody Picoband®
Cat # A00962-2
Our recommended anti-FOXC1 antibody is a best-performing, extensively cited reagent, rigorously validated by western blot and orthogonally cross-validated against negative-control tissues and complementary detection methods, ensuring specific, reproducible detection of FOXC1 across experimental systems.
Which to pick: Only one Boster FOXC1 antibody is catalogued, A00962-2, which includes an actual western blot validation image demonstrating specific detection—making it the clear default choice for your FOXC1 western blot experiments.
Source: BosterBio FOXC1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q12948.
- Human Protein Atlas. FOXC1 tissue expression.