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- Table of Contents
FOXC2 / Forkhead box protein C2 · Western blot design guide
Design a Western Blot for FOXC2
Real validated FOXC2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-FOXC2 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
FOXC2 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 | ~53.7 kDa | |
| Observed band | Approximately 60 kDa | |
| Gel | 10% (catalog A01780-2) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Methylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated FOXC2 Western Blot Protocols
The A01780-2 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, human A375 (catalog A01780-2) |
| Gel % | 10% (catalog A01780-2) |
| Load | 30 ug; reducing conditions (catalog A01780-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01780-2) |
| Membrane | nitrocellulose membrane (catalog A01780-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01780-2) |
| Primary antibody | A01780-2 · 0.5 μg/mL (catalog A01780-2) |
| Primary incubation | overnight at 4°C (catalog A01780-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01780-2) |
| Secondary incubation | 1.5 hour at RT (catalog A01780-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01780-2) |
| Detection | ECL (catalog A01780-2) |
Section 2
What Is the Expected FOXC2 Western Blot Band Size?
FOXC2 predicts at 53.7 kDa but is observed near 60 kDa; the cause of this difference is not established.
| Band near 60 kDa | Empirical FOXC2 band; confirm identity with antibody controls |
| Band near 53.7 kDa | Near the predicted FOXC2 sequence mass; confirm identity |
| Closely spaced bands | Potentially different phosphorylation states; separation is not established |
| Additional higher band | Possible ubiquitin-like conjugated FOXC2; confirm identity |
💡Expected FOXC2 appearanceFOXC2 has a predicted mass of 53.7 kDa and an empirical band near 60 kDa in reducing whole-cell blots; the difference is unexplained, so confirm band identity with ordinary controls.
| Predicted FOXC2 mass | 53.7 kDa from the protein sequence; an empirical band appears near 60 kDa |
| Phosphoserine sites including Ser215 | Phosphorylation may alter mobility, but a visible shift is not established |
| Phosphothreonine at Thr247 | Phosphorylation may alter mobility, but a visible shift is not established |
| Omega-N-methylarginine at Arg279 | Methylation is listed; its effect on band position is not established |
| Ubiquitin-like conjugation | Could produce a higher band if present; no band size is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear FOXC2 may be poorly recovered | Check nuclear extraction and a positive-control lysate |
| Band higher than expected | FOXC2 is observed near 60 kDa despite a 53.7 kDa predicted mass; the cause is unknown | Compare with a positive control and verify antibody specificity |
| Band lower than expected | Identity or sample integrity is uncertain | Check sample integrity and confirm with a second FOXC2 antibody |
| Multiple bands | Phosphorylation states are possible, but distinct bands are unproven | Compare phosphatase-treated and untreated samples and verify band identity |
| Weak or no signal | Nuclear FOXC2 may be insufficiently recovered | Use a nuclear-enriched sample and a positive control |
| Fragments below expected size | Sample degradation or nonspecific binding is possible | Check sample integrity and confirm with a second FOXC2 antibody |
Experiment evidence
Western blot images showing FOXC2 band
A01780-2 · FOXC2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for FOXC2 Western blot
🧪For positive controls for FOXC2 in Western blot, you can use an HPA-IHC candidate positive sample once one is identified; none is provided in the supplied evidence.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: FOXC2 is nuclear, but the supplied HPA data identify no tissue controls.
HPA tissue expression evidence for FOXC2
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced FOXC2 Western Blot Tips
Deeper troubleshooting and optimisation questions for FOXC2, answered from its protein features.
How should FOXC2 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 FOXC2 isoforms explain multiple bands?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It does not support assigning additional bands to known FOXC2 isoforms. Check band identity with an appropriate FOXC2 control.
Which FOXC2 phosphorylation sites matter when interpreting bands?
PTM · UniProt annotates phosphoserine at positions 215, 219, 232, 235, 240, 251, 281, 288, and 367, plus phosphothreonine at 247. These are UniProt coordinates; antibody or paper numbering may differ. Their presence does not establish a visible band shift.
Does this guide establish induction of FOXC2?
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 FOXC2 Western blot?
Transfer · FOXC2 is predicted at 53.7 kDa and has been observed near 60 kDa. Use transfer conditions suitable for a protein in that size range, then check transfer efficiency around 60 kDa on the membrane and gel.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A01780-2 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.
Which fraction is most relevant for FOXC2 quantitation?
Quantitation · UniProt places FOXC2 in the nucleus. For comparisons across samples, measure the same fraction consistently and use a loading control appropriate to that fraction.
Why might FOXC2 appear near 60 kDa instead of 53.7 kDa?
Interpretation · FOXC2 has a predicted mass of 53.7 kDa and an observed band near 60 kDa. UniProt lists phosphorylation and arginine methylation, but those annotations alone do not establish the cause of the apparent mass difference. Compare the band with a molecular weight marker and an appropriate FOXC2 control.
UniProt lists omega-N-methylarginine at positions 279 and 293 and asymmetric dimethylarginine at 458. These are UniProt coordinates; check the numbering convention before interpreting a site-specific antibody. The annotations alone do not predict a separate band.
Start with the expected approximately 60 kDa band and test other bands with an appropriate FOXC2 control. The record lists one isoform, phosphorylation, and methylation, but does not establish the identity of additional bands or show that a modification causes a visible shift.
UniProt lists omega-N-methylarginine at positions 279 and 293 and asymmetric dimethylarginine at 458. These are UniProt coordinates; check the numbering convention before interpreting a site-specific antibody. The annotations alone do not predict a separate band.
Start with the expected approximately 60 kDa band and test other bands with an appropriate FOXC2 control. The record lists one isoform, phosphorylation, and methylation, but does not establish the identity of additional bands or show that a modification causes a visible shift.
Boster reagents
FOXC2 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-FOXC2 Picoband® Antibody
Cat # A01780-2
A01780-2 is a rabbit polyclonal anti-FOXC2 antibody with a Western blot image showing an approximately 60 kDa band in tested human, rat, and mouse cell lysates. The expected FOXC2 size is 54 kDa; no additional validation evidence is supplied.
Which to pick: A01780-2 is the only listed option. Its Western blot image includes human HeLa, 293T, A375, and PC-3; rat PC-12 and NRK; and mouse C2C12 and 3T3-L1 lysates. Choose it based on the reported reactivity and these tested sample contexts.
Source: BosterBio FOXC2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q99958.
- Human Protein Atlas. FOXC2 tissue expression.
- PMC6894326 — target-verified WB comparison
- PMC7249675 — target-verified WB comparison
- PMC7418171 — target-verified WB comparison