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- Table of Contents
GATA4 / Transcription factor GATA-4 · Western blot design guide
Design a Western Blot for GATA4
Real validated GATA4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-GATA4 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
GATA4 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 | ~44.6 kDa | |
| Observed band | ~54 kDa | |
| Gel | 10% (catalog A00499-1) | |
| Positive control | Esophagus (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 | Methylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated GATA4 Western Blot Protocols
The A00499-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HepG2, human PC-3, human HUH7 (catalog A00499-1) |
| Gel % | 10% (catalog A00499-1) |
| Load | 30 ug; reducing conditions (catalog A00499-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00499-1) |
| Membrane | nitrocellulose membrane (catalog A00499-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00499-1) |
| Primary antibody | A00499-1 · 0.5 μg/mL (catalog A00499-1) |
| Primary incubation | overnight at 4°C (catalog A00499-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00499-1) |
| Secondary incubation | 1.5 hour at RT (catalog A00499-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00499-1) |
| Detection | ECL (catalog A00499-1) |
Section 2
What Is the Expected GATA4 Western Blot Band Size?
GATA4 is predicted at 44.6 kDa and observed at ~54 kDa in antibody QC blots; the cause of the difference is not established.
| Band near 54 kDa | Empirical GATA4 band in reducing whole-cell lysates; its difference from the predicted mass is unexplained |
| Band near 44.6 kDa | Candidate band near the UniProt predicted mass; confirm its identity |
| Multiple bands | Isoforms 1 and 2 are annotated, but their migration and resolution are unknown |
| Little or no band in a cytosolic fraction | GATA4 is nuclear |
💡Expected GATA4 appearanceUniProt predicts 44.6 kDa, while antibody QC reports a ~54 kDa band in reducing whole-cell lysates; the difference is unexplained, so confirm band identity with positive and knockdown controls.
| UniProt predicted mass | 44.6 kDa is the sequence-based estimate; the observed band is ~54 kDa |
| Alternative splicing | Can alter protein length, but no isoform masses or migration differences are supplied |
| Isoform 1 | Its size relative to isoform 2 is unspecified |
| Isoform 2 | Its size relative to isoform 1 is unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear GATA4 may be poorly recovered | Check a nuclear fraction alongside a positive whole-cell lysate |
| Band higher than expected | The reported ~54 kDa band exceeds the 44.6 kDa prediction for an unknown reason | Check the molecular-weight marker and confirm identity by GATA4 knockdown |
| Band lower than expected | An alternative isoform is possible, but its migration is unreported | Confirm identity with GATA4 knockdown and an antibody to another epitope |
| Multiple bands | Isoforms 1 and 2 are annotated, but distinct bands are not established | Test which bands decrease after GATA4 knockdown |
| Weak or no signal | Nuclear GATA4 may be dilute in whole-cell lysate | Try nuclear enrichment and include a positive lysate |
Experiment evidence
Western blot images showing GATA4 band
A00499-1 · GATA4
M00499-1 · GATA4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for GATA4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for GATA4 in Western blot, you can use esophagus, which HPA scores High for GATA4 expression.
Positive control: Esophagus (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: GATA4 is nuclear, so nuclear-enriched lysates may improve detection.
HPA tissue expression evidence for GATA4
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 |
|---|---|---|---|---|
| Esophagus | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | High | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | High | Protein (IHC) | HPA → |
| Placenta | trophoblastic cells | High | Protein (IHC) | HPA → |
| Small intestine | glandular cells | High | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced GATA4 Western Blot Tips
Deeper troubleshooting and optimisation questions for GATA4, answered from its protein features.
How should GATA4 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 GATA4 isoforms produce separate bands?
Isoforms · UniProt lists isoforms 1 and 2. At canonical position 205, isoform 2 has LV in place of L, adding one residue. This small sequence difference does not establish that the isoforms resolve as separate bands.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of GATA4?
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 GATA4?
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 A00499-1 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.
What matters when quantifying GATA4 Western blot signal?
Quantitation · GATA4 is listed in the nucleus. Compare samples prepared with the same fractionation approach and normalize signals within that approach. If isoforms produce distinguishable signals, specify which band is quantified; the supplied features do not establish that they separate.
Why might GATA4 appear near 54 kDa instead of 44.6 kDa?
Interpretation · The supplied observed band is approximately 54 kDa, while the predicted mass is 44.6 kDa. The listed features do not establish the cause of this difference. Use the observed position as a reference, but confirm band identity independently.
UniProt lists N6-methyllysine at position 300, modified by EZH2. Position 300 uses the supplied UniProt coordinate convention; numbering in other sources may differ. This modification alone does not demonstrate a visible shift or explain the approximately 54 kDa band.
Isoform 2 changes canonical residue 205 from L to LV. If an antibody recognizes a sequence spanning that position, check its stated epitope and isoform coverage before interpreting unequal signals. The supplied features do not establish antibody specificity.
Compare them with the approximately 54 kDa observed band and consider the two listed isoforms. The supplied features list no glycosylation sites, signal peptide, or propeptide, so they provide no basis for assigning extra bands to those processes. Band position alone does not identify a band.
UniProt lists N6-methyllysine at position 300, modified by EZH2. Position 300 uses the supplied UniProt coordinate convention; numbering in other sources may differ. This modification alone does not demonstrate a visible shift or explain the approximately 54 kDa band.
Isoform 2 changes canonical residue 205 from L to LV. If an antibody recognizes a sequence spanning that position, check its stated epitope and isoform coverage before interpreting unequal signals. The supplied features do not establish antibody specificity.
Compare them with the approximately 54 kDa observed band and consider the two listed isoforms. The supplied features list no glycosylation sites, signal peptide, or propeptide, so they provide no basis for assigning extra bands to those processes. Band position alone does not identify a band.
Boster reagents
GATA4 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-GATA4 Antibody Picoband®
Cat # A00499-1
Real WB data
Anti-GATA4 Rabbit Monoclonal Antibody
Cat # M00499-1
Both listed anti-GATA4 antibodies have Western blot images showing a band near 54 kDa, versus the stated 45 kDa expectation. Captions show specific human cell lines and rat liver; M00499-1 also shows mouse liver. These examples do not establish universal sample performance.
Which to pick: For mouse liver, M00499-1 has a matching image and lists mouse reactivity. Both antibodies have images for human HepG2 cells and rat liver. A00499-1 also shows PC-3, HUH7, and 293T cells; M00499-1 also shows SH-SY5Y cells.
Source: BosterBio GATA4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P43694.
- Human Protein Atlas. GATA4 tissue expression.
- PMC3135351 — target-verified WB comparison
- PMC10696868 — target-verified WB comparison
- PMC5931571 — target-verified WB comparison