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- Table of Contents
TBX3 / T-box transcription factor TBX3 · Western blot design guide
Design a Western Blot for TBX3
Real validated TBX3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TBX3 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
TBX3 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 | ~79.4 kDa | |
| Gel | 12–15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated TBX3 Western Blot Protocols
The M01107-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | MCF7 cell lysate (catalog M01107-1) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M01107-1; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected TBX3 Western Blot Band Size?
TBX3 is predicted at 79.4 kDa; isoforms and phosphorylation could affect migration, but no distinct band position is demonstrated.
| Band near 79.4 kDa | Consistent with the predicted TBX3 mass; identity requires validation |
| Several bands at different positions | Could reflect isoforms I, II, III, and IV; their migration is unknown |
| Close doublet near the predicted size | Could reflect phosphorylation at annotated serines; a visible shift is unproven |
| Stronger band in nuclear extract | Consistent with TBX3's nuclear localization |
💡Expected TBX3 appearanceUniProt predicts 79.4 kDa for TBX3; no empirical band is supplied, and the annotated isoforms and phosphorylation sites do not establish distinct migration, so confirm band identity with appropriate controls.
| Predicted TBX3 mass | Places the reference size at 79.4 kDa |
| Isoform I | May migrate differently from other isoforms; its mass is unspecified |
| Isoform II | May migrate differently from other isoforms; its mass is unspecified |
| Isoform III | May migrate differently from other isoforms; its mass is unspecified |
| Isoform IV | May migrate differently from other isoforms; its mass is unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear TBX3 may be insufficiently represented in the sample | Check loading and test a nuclear extract with a validated positive control |
| Band higher than expected | An isoform or phosphorylation could affect migration, but neither shift is established | Compare a validated positive control and confirm identity with TBX3 depletion |
| Band lower than expected | An isoform or sample degradation could produce a smaller band | Use fresh lysate with protease inhibitors and confirm identity with TBX3 depletion |
| Multiple bands | Annotated isoforms are possible, but nonspecific binding is also possible | Compare isoform controls and test whether bands disappear after TBX3 depletion |
| Weak or no signal | TBX3 signal may be low in the tested sample | Check loading, use a validated positive control, and assess a nuclear extract |
Experiment evidence
Western blot images showing TBX3 band
M01107-1 · TBX3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TBX3 Western blot
🧪For positive controls for TBX3 in Western blot, you can use no HPA-supported tissue or cell sample from 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: With no HPA expression data, a reliable positive sample or negative tissue cannot be identified.
HPA tissue expression evidence for TBX3
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 TBX3 Western Blot Tips
Deeper troubleshooting and optimisation questions for TBX3, answered from its protein features.
How should TBX3 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.
How could TBX3 isoforms affect band interpretation?
Isoforms · Four isoforms are listed: II, I, III, and IV. Relative to the supplied sequence, isoform I lacks residues 221–240; isoform III lacks 490–615 and 661–677. These sequence differences could affect migration or antibody recognition. No sequence change is supplied for isoform IV, so its band position cannot be predicted here.
Which TBX3 phosphorylation sites are annotated?
PTM · UniProt lists phosphoserines at positions 371, 432, 438, 456, 707, 738, 740, and 742. These are UniProt sequence coordinates; paper or antibody numbering may differ. Their annotation alone does not show that a separate band will appear.
If bands differ between samples, consider the eight annotated phosphoserines as possible contributors, then test phosphorylation directly before assigning a band. A band difference by itself cannot identify a modified site or establish that phosphorylation caused the difference.
If bands differ between samples, consider the eight annotated phosphoserines as possible contributors, then test phosphorylation directly before assigning a band. A band difference by itself cannot identify a modified site or establish that phosphorylation caused the difference.
Does this guide establish induction of TBX3?
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.
How should transfer be checked for TBX3?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M01107-1 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 TBX3 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Should TBX3 migrate at exactly 79.4 kDa?
Interpretation · 79.4 kDa is the supplied predicted mass, and no observed band position is available. The listed isoforms and phosphorylation sites are useful when investigating a different apparent mass, but their presence alone does not establish a visible shift.
TBX3 is annotated as nuclear. Check the nuclear fraction when assessing its detection, and account for fraction differences when comparing band intensity between samples.
Compare them with the 79.4 kDa prediction and consider the documented isoform sequence differences and phosphoserines. Check whether the antibody can recognize the relevant isoforms, especially if its target region overlaps a deleted segment. The supplied features do not establish the identity of any unexpected band.
TBX3 is annotated as nuclear. Check the nuclear fraction when assessing its detection, and account for fraction differences when comparing band intensity between samples.
Compare them with the 79.4 kDa prediction and consider the documented isoform sequence differences and phosphoserines. Check whether the antibody can recognize the relevant isoforms, especially if its target region overlaps a deleted segment. The supplied features do not establish the identity of any unexpected band.
Boster reagents
TBX3 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-Tbx3 Rabbit Monoclonal Antibody
Cat # M01107-1
The catalog reports one anti-TBX3 antibody for Western blot: M01107-1, a rabbit monoclonal reported to react with human, mouse, and rat. Its WB image shows Tbx3 expression in MCF7 cell lysate; the supplied evidence does not show other tested samples.
Which to pick: M01107-1 is the only listed option and has a WB image using MCF7 cell lysate. Check its stated human, mouse, and rat reactivity against your sample; the image documents the MCF7 context only.
Source: BosterBio TBX3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O15119.
- Human Protein Atlas. TBX3 tissue expression.
- PMC8813229 — target-verified WB comparison
- PMC4865973 — target-verified WB comparison
- PMC6320639 — target-verified WB comparison