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- Table of Contents
STAT4 / Signal transducer and activator of transcription 4 · Western blot design guide
Design a Western Blot for STAT4
Real validated STAT4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-STAT4 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
STAT4 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 | ~85.9 kDa | |
| Observed band | ~86 kDa | |
| Gel | 10% (catalog PA1692) | |
| 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 + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated STAT4 Western Blot Protocols
The PA1692 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A549, human Jurkat, human HEL (catalog PA1692) |
| Gel % | 10% (catalog PA1692) |
| Load | 30 ug; reducing conditions (catalog PA1692) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog PA1692) |
| Membrane | nitrocellulose membrane (catalog PA1692) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog PA1692) |
| Primary antibody | PA1692 · 0.5 μg/mL (catalog PA1692) |
| Primary incubation | overnight at 4°C (catalog PA1692) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog PA1692) |
| Secondary incubation | 1.5 hour at RT (catalog PA1692) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog PA1692) |
| Detection | ECL (catalog PA1692) |
Section 2
What Is the Expected STAT4 Western Blot Band Size?
STAT4 is predicted at 85.9 kDa and observed at ~86 kDa; the supplied evidence does not establish a cause for the small difference.
| Band near 86 kDa | Matches the observed STAT4 band and its predicted monomer mass. |
| Band near 172 kDa | Could reflect a preserved STAT4 homodimer; confirm its identity. |
| Stronger nuclear STAT4 band after stimulation | Consistent with phosphorylation responsive nuclear translocation. |
| Weaker cytoplasmic STAT4 band after stimulation | Could accompany redistribution of STAT4 to the nucleus. |
💡Expected STAT4 appearanceSTAT4 has a predicted mass of 85.9 kDa and an empirical band at ~86 kDa; confirm band identity with appropriate antibody and sample controls.
| Predicted STAT4 mass | Predicts an 85.9 kDa monomer, consistent with the observed ~86 kDa band. |
| STAT4 homodimer formation | Could yield a band near twice monomer mass if the dimer survives sample preparation. |
| JAK phosphorylation at Tyr693 | May affect mobility, but no visible size shift is established. |
| MAP2K6 phosphorylation at Ser721 | May affect mobility, but no visible size shift is established. |
| Acetylation at Lys667 | Is documented, but no visible size shift is established. |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | A preserved STAT4 homodimer is possible. | Compare denaturing and reducing preparations and confirm band identity. |
| Band lower than expected | The supplied features do not establish a smaller STAT4 form. | Check the molecular weight marker and validate with a second STAT4 antibody. |
| Multiple bands | STAT4 modifications are documented, but distinct bands are not established. | Compare phosphatase treated samples and check antibody specificity. |
| Weak or no signal | STAT4 can redistribute between cytoplasm and nucleus after phosphorylation. | Check whole cell and nuclear fractions with loading and fraction controls. |
| Fragments below expected size | The supplied features do not identify STAT4 cleavage products. | Check sample integrity and confirm fragments with a second STAT4 antibody. |
Experiment evidence
Western blot images showing STAT4 band
PA1692 · STAT4
M00734 · STAT4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for STAT4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for STAT4 in Western blot, you can use testis tissue, which HPA rates High.
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 alongside the samples.
⚠️Feasibility: Tissue controls are feasible, though phosphorylation can shift STAT4 from cytoplasm to nucleus.
HPA tissue expression evidence for STAT4
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 | round or early spermatids | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | Medium | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | Medium | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Small intestine | glandular 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 → |
| Bronchus | basal cells | Not detected | Protein (IHC) | HPA → |
| Cervix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced STAT4 Western Blot Tips
Deeper troubleshooting and optimisation questions for STAT4, answered from its protein features.
How should STAT4 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.
Are multiple STAT4 isoform bands expected?
Isoforms · The supplied features list one isoform and no alternative sequence. They do not support assigning an additional band to a named STAT4 isoform.
Which STAT4 phosphorylation sites can I measure?
PTM · UniProt lists Tyr693 phosphorylation by JAK and Ser721 phosphorylation by MAP2K6. Use site-specific detection to distinguish these signals, and report positions using UniProt numbering if an antibody uses another convention.
Measure the phospho-specific signal alongside total STAT4 in comparable samples. State whether the assay detects UniProt Tyr693 or Ser721, since the listed enzymes and sites differ.
UniProt lists N6-acetyllysine at Lys667. This establishes a modification site, but does not show that acetylation produces a visible band shift or changes the observed mass near 86 kDa.
Measure the phospho-specific signal alongside total STAT4 in comparable samples. State whether the assay detects UniProt Tyr693 or Ser721, since the listed enzymes and sites differ.
UniProt lists N6-acetyllysine at Lys667. This establishes a modification site, but does not show that acetylation produces a visible band shift or changes the observed mass near 86 kDa.
Does this guide establish induction of STAT4?
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 STAT4?
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 PA1692 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 STAT4 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.
Why is the STAT4 band near 86 kDa?
Interpretation · The observed band near 86 kDa closely matches STAT4’s predicted mass of 85.9 kDa. The listed modifications do not establish a visible mobility shift.
STAT4 is listed in both cytoplasm and nucleus, with nuclear translocation in response to phosphorylation. Compare nuclear and cytoplasmic signals when examining a phosphorylation response; localization alone does not identify which site is modified.
Compare it with the expected band near 86 kDa. The supplied features list no signal peptide, propeptide, glycosylation sites, or alternative sequence to explain another band. Phosphorylation and acetylation are listed, but their presence alone cannot establish the cause of a separate band.
STAT4 is listed in both cytoplasm and nucleus, with nuclear translocation in response to phosphorylation. Compare nuclear and cytoplasmic signals when examining a phosphorylation response; localization alone does not identify which site is modified.
Compare it with the expected band near 86 kDa. The supplied features list no signal peptide, propeptide, glycosylation sites, or alternative sequence to explain another band. Phosphorylation and acetylation are listed, but their presence alone cannot establish the cause of a separate band.
Boster reagents
STAT4 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-STAT4 Antibody Picoband®
Cat # PA1692
Real WB data
Anti-STAT4 Rabbit Monoclonal Antibody
Cat # M00734
Two the supplier anti-STAT4 antibodies have WB images. PA1692 shows an approximately 86 kDa band in the listed human cell, rat testis, and mouse testis and thymus lysates. M00734 shows STAT4 expression in HeLa lysate; its caption gives no band size or protocol details.
Which to pick: Choose PA1692 for its stated human, rat, and mouse reactivity and detailed WB example. M00734 lists human reactivity and a HeLa WB image, but its caption provides fewer experimental details. These are the only two listed options.
Source: BosterBio STAT4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q14765.
- Human Protein Atlas. STAT4 tissue expression.
- PMC11989168 — target-verified WB comparison
- PMC11087736 — target-verified WB comparison
- PMC8826371 — target-verified WB comparison