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- Table of Contents
TCF4 / Transcription factor 4 · Western blot design guide
Design a Western Blot for TCF4
Real validated TCF4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TCF4 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
TCF4 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 | ~71.3 kDa | |
| Observed band | ~71 kDa | |
| Gel | 5–20% (catalog A00674-2) | |
| Positive control | Testis (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
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 | 16 isoform(s) |
Section 1
Real Curated TCF4 Western Blot Protocols
The A00674-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human SH-SY5Y, human Daudi, rat testis (catalog A00674-2) |
| Gel % | 5–20% (catalog A00674-2) |
| Load | 30 ug; reducing conditions (catalog A00674-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00674-2) |
| Membrane | nitrocellulose membrane (catalog A00674-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00674-2) |
| Primary antibody | A00674-2 · 0.5 μg/mL (catalog A00674-2) |
| Primary incubation | overnight at 4°C (catalog A00674-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00674-2) |
| Secondary incubation | 1.5 hour at RT (catalog A00674-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00674-2) |
| Detection | ECL (catalog A00674-2) |
Section 2
What Is the Expected TCF4 Western Blot Band Size?
TCF4 is predicted at 71.3 kDa and observed at ~71 kDa; the cause of the small difference is not established.
| Band at ~71 kDa | matches the reported TCF4 band and its 71.3 kDa predicted mass |
| Additional discrete bands | could include TCF4 splice isoforms, but their migration is unknown |
| Stronger ~71 kDa band in nuclear extract | consistent with TCF4's nuclear localization |
| Mobility change after phosphatase treatment | could reflect phosphorylation at documented serine sites |
💡Expected TCF4 appearanceTCF4 has a predicted mass of 71.3 kDa and an empirical band at ~71 kDa; confirm band identity with knockdown or a second antibody before assigning other bands.
| Predicted TCF4 mass | 71.3 kDa provides the reference for the observed ~71 kDa band |
| SEF2-1B, SEF2-1A, SEF2-1D, and B+delta isoforms | may differ in size; individual masses and band separation are unknown |
| B-delta, A-, G-, H-, D-, and F- isoforms | may differ in size; individual masses and band separation are unknown |
| 11, E-, 13, C-, C-delta, and I- isoforms | may differ in size; individual masses and band separation are unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear TCF4 may be poorly recovered or too dilute in the preparation | Check nuclear extraction and include a known positive lysate |
| Band higher than expected | An isoform or phosphorylation is possible, but neither explains a specific shift here | Compare with the ~71 kDa band using TCF4 knockdown or a second antibody |
| Band lower than expected | An isoform or degradation is possible; its identity is unverified | Use fresh, protease-inhibited lysate and a TCF4 knockdown control |
| Multiple bands | TCF4 has multiple splice isoforms, though distinct bands are not established | Check which bands respond to TCF4 knockdown |
| Weak or no signal | TCF4 recovery or abundance may be low in the tested sample | Check a positive lysate and enrich the nuclear fraction |
| Fragments below expected size | Proteolysis during sample preparation is possible | Prepare fresh lysate with protease inhibitors and verify band identity |
Experiment evidence
Western blot images showing TCF4 band
A00674-2 · TCF4
M00674 · TCF4
A00674S616 · TCF4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TCF4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TCF4 in Western blot, you can use testis lysate, which has high HPA expression.
Positive control: Testis (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Because TCF4 is nuclear, nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for TCF4
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 |
|---|---|---|---|---|
| Testis | pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | adipocytes | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Nasopharynx | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TCF4 Western Blot Tips
Deeper troubleshooting and optimisation questions for TCF4, answered from its protein features.
How should TCF4 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 TCF4 isoforms produce different bands?
Isoforms · Yes. UniProt lists 16 isoforms with sequence changes. For example, I- lacks residues 1–216, SEF2-1A lacks 1–160, and D- lacks 1–130. These differences could affect apparent band patterns, but the features alone do not predict exact band positions.
Map the antibody epitope against the isoform sequences. Several isoforms alter or remove N-terminal regions, while B+delta, SEF2-1A, SEF2-1D, 11, and 13 replace residue 545 with TRSRS. An antibody targeting an altered region may detect only some isoforms.
Decide whether to quantify one band or the combined TCF4 signal, and use the same approach across samples. Because 16 isoforms are listed, confirm which forms the antibody recognizes before interpreting a change in signal as a change in total TCF4.
Map the antibody epitope against the isoform sequences. Several isoforms alter or remove N-terminal regions, while B+delta, SEF2-1A, SEF2-1D, 11, and 13 replace residue 545 with TRSRS. An antibody targeting an altered region may detect only some isoforms.
Decide whether to quantify one band or the combined TCF4 signal, and use the same approach across samples. Because 16 isoforms are listed, confirm which forms the antibody recognizes before interpreting a change in signal as a change in total TCF4.
Which TCF4 phosphorylation sites matter when interpreting bands?
PTM · UniProt lists phosphoserines at positions 66, 87, 92, 372, and 515 in its reference sequence. Check the isoform and numbering convention used by the antibody, since isoform sequence changes can alter corresponding positions. These annotations alone do not establish a visible band shift.
Does this guide establish induction of TCF4?
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 TCF4?
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 A00674-2 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.
How should TCF4 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.
How does the observed TCF4 band compare with predicted mass?
Interpretation · The reported band near 71 kDa agrees with the 71.3 kDa predicted mass. The listed features do not establish a measurable mass shift; compare any additional bands with the documented isoforms before assigning them.
Compare their positions with the expected near-71 kDa band and check antibody coverage of documented isoforms. N-terminal deletions, other sequence changes, and annotated phosphorylation are possible considerations, but the supplied features cannot identify an unexpected band by position alone.
TCF4 is annotated as nuclear. Comparing nuclear-enriched and whole-cell samples can help assess whether sample composition affects the detected signal. Interpret that comparison alongside antibody recognition of the documented isoforms.
Compare their positions with the expected near-71 kDa band and check antibody coverage of documented isoforms. N-terminal deletions, other sequence changes, and annotated phosphorylation are possible considerations, but the supplied features cannot identify an unexpected band by position alone.
TCF4 is annotated as nuclear. Comparing nuclear-enriched and whole-cell samples can help assess whether sample composition affects the detected signal. Interpret that comparison alongside antibody recognition of the documented isoforms.
Boster reagents
TCF4 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-TCF4 Antibody Picoband®
Cat # A00674-2
Real WB data
Anti-TCF4 Rabbit Monoclonal Antibody
Cat # M00674
Real WB data
Anti-TCF-4/12 (S616) Antibody
Cat # A00674S616
All three listed antibodies have WB images. A00674-2 reports a band near 71 kDa in human, rat, and mouse samples under stated conditions. M00674 shows SH-SY5Y lysate; A00674S616 shows six lysates at 1:500, without a reported band size.
Which to pick: For mouse or rat samples, A00674-2 has the clearest documented WB result, including a reported band size. For human SH-SY5Y lysate, M00674 also has a WB image. A00674S616 offers additional lysate examples; its caption gives less detail about the result.
Source: BosterBio TCF4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P15884.
- Human Protein Atlas. TCF4 tissue expression.
- PMC9666405 — target-verified WB comparison
- PMC12571441 — target-verified WB comparison
- PMC7401711 — target-verified WB comparison