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GATA5 · Western blot design guide
Design a Western Blot for GATA5
Real validated GATA5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-GATA5 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
GATA5 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 | ~41.3 kDa | |
| Observed band | ~50 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | — | |
| Regulation | LPS-induced | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated GATA5 Western Blot Protocols
Literature-validated Western blot parameters for GATA5 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human 293T , Lane 2: human HepG2 . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-GATA5 antigen affinity purified polyclonal antibody (Catalog # PB9200) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for GATA5 at approximately 50 kDa. The expected band size for GATA5 is at 41 kDa |
| Gel % | 10–12% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 50 kDa |
Section 2
What Is the Expected GATA5 Western Blot Band Size?
GATA5 has a 41.3 kDa calculated backbone but typically runs near 50 kDa on Western blot, likely from anomalous SDS-PAGE migration of its zinc-finger DNA-binding domains.
| single band near 50 kDa | represents full-length GATA5 migrating slower than its 41.3 kDa calculated mass, consistent with the empirically observed band |
| no smear or ladder above the main band | GATA5 has no annotated glycosylation sites, so glycoform-driven smearing is not expected |
| no doublet near ~80-100 kDa | no inter-chain disulfide bonds are annotated, so a covalent SDS-resistant homodimer is not expected |
| single band without extra splice-variant bands | only one isoform is annotated, so additional bands are not expected from alternative splicing |
| stronger signal in nuclear-enriched preps than whole-cell lysate | GATA5 is annotated as a nuclear protein, so signal concentrates in the nuclear fraction |
💡Expected GATA5 appearanceExpect a single band at approximately 50 kDa, higher than the 41.3 kDa calculated mass, consistent with the anomalous SDS-PAGE migration typical of zinc-finger DNA-binding transcription factors like GATA5.
| Predicted mass (UniProt) | calculated at 41.3 kDa from the 397-aa sequence, the baseline against which the observed band is compared |
| Zinc-finger/DNA-binding domain composition | basic, structured zinc-finger and DNA-binding regions can reduce SDS binding and slow migration, producing an apparent band around 50 kDa above the calculated mass |
| No annotated glycosylation sites | rules out glycosylation as a cause of the upward mass shift or of smearing |
| No annotated disulfide bonds or oligomer state | rules out covalent homodimer formation as a cause of a higher-molecular-weight band |
| Single annotated isoform | no alternative-splicing-driven band-size variation is expected |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | zinc-finger DNA-binding transcription factors like GATA5 often migrate anomalously slower on SDS-PAGE, giving an apparent ~50 kDa band versus the 41.3 kDa calculated mass | confirm identity with a knockdown or overexpression control and treat ~50 kDa as the expected native band rather than assuming degradation |
| No band in lysate | GATA5 is a low-abundance nuclear transcription factor, so whole-cell lysate without nuclear enrichment can dilute signal below detection | use a nuclear or nuclear-enriched extraction protocol and confirm GATA5 expression in the cell line before blotting |
| Multiple bands | only one isoform is annotated for GATA5, so extra bands are more likely nonspecific antibody binding or degradation than genuine splice variants | validate specificity with a knockdown/knockout or overexpression control and confirm which band matches the expected ~50 kDa size |
| Weak or no signal | low endogenous expression of this nuclear transcription factor in many cell types can yield weak signal | increase loading amount, use nuclear extraction, or test a cell line/tissue with known GATA5 expression |
| Fragments below expected size | degradation of the transcription factor during lysis can generate lower-molecular-weight fragments | add protease inhibitors during lysis, keep samples cold, and minimize freeze-thaw cycles |
| Broad smear instead of sharp band | GATA5 has no annotated glycosylation sites, so a broad smear is not expected from glycoform heterogeneity and more likely reflects overloading or poor transfer | reduce sample loading, optimize transfer conditions, and ensure complete denaturation before loading |
Experiment evidence
Western blot images showing GATA5 band
PB9200 · GATA5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for GATA5 Western blot
🧪For positive controls for GATA5 in Western blot, you can use lysates from cells transiently transfected with a GATA5 expression construct, since the Human Protein Atlas has no tissue or cell line expression data on record for this protein.
Positive control: GATA5-transfected/overexpressing cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Include GAPDH and β-actin as loading controls alongside a total-protein stain (e.g., stain-free imaging, Ponceau S, or REVERT) to confirm even loading and transfer.
⚠️Feasibility: Because no HPA expression data exist for GATA5, a validated endogenous positive tissue or a clean negative tissue cannot be identified from current evidence, so specificity should be confirmed with an overexpression positive control and an siRNA knockdown or CRISPR KO negative control.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced GATA5 Western Blot Tips
Deeper troubleshooting and optimisation questions for GATA5, answered from its protein features.
Why does GATA5 run heavier than its predicted 41 kDa mass?
GATA5's zinc-finger DNA-binding domains carry a high density of basic and cysteine residues that bind SDS less efficiently, slowing migration. This commonly shifts zinc-finger transcription factors to an apparent mass several kDa above the calculated 41.3 kDa, so the ~50 kDa band seen on blots is consistent with the sequence rather than evidence of a modified or alternate species.
Why does GATA5 signal vary across cell lines or tissues?
GATA5 is annotated as a transcriptional activator confined to the nucleus, so its abundance tracks the cell's differentiation and transcriptional state rather than being constitutively expressed. Weak or absent signal in a given lysate may simply reflect low endogenous expression in that cell type; enriching for nuclear fractions before loading improves detection of this low-abundance regulator.
Should extra bands be expected from post-translational modification?
UniProt lists no modified residues, glycosylation, or disulfide bonds for GATA5, so a single unmodified species around 41-50 kDa is expected. Additional bands are more likely degradation fragments or nonspecific antibody binding than PTM-driven isoforms, since no PTM sites are annotated for this protein.
What blocking buffer works best for GATA5 detection?
Because GATA5 is a metal-binding zinc-finger protein, use BSA rather than milk-based blockers, which contain phosphoproteins and metal-binding caseins that can raise background on metal-binding targets. 5% BSA in TBST gives cleaner blocking for this nuclear DNA-binding factor without competing for zinc-dependent epitopes.
What transfer method to use for GATA5 Western blot?
At ~41-50 kDa, standard wet or semi-dry transfer onto PVDF works well for GATA5. Because it is a nuclear DNA-binding protein, ensure lysates are fully denatured and sonicated to disrupt chromatin association before loading, as incomplete extraction from the nucleus can trap the protein and reduce transfer efficiency.
Which loading control suits GATA5 quantitation best?
Since GATA5 is nuclear-localized, normalize to a nuclear loading control such as Lamin B1, TBP, or Histone H3 rather than cytoplasmic GAPDH or beta-actin. With only one annotated isoform, a single specific band should be used for densitometry, simplifying quantitation compared to multi-isoform GATA family members.
What explains unexpected bands near the GATA5 signal?
GATA5 shares a conserved zinc-finger repeat domain with other GATA family members, so antibodies can cross-react with GATA4 or GATA6 at similar molecular weights. Faint lower-molecular-weight bands likely reflect proteolytic degradation of the zinc-finger regions during nuclear lysate preparation rather than genuine alternate isoforms, since only one isoform is annotated.
Boster reagents
Best GATA5 Western Blot Antibodies
BosterBio's GATA5 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-GATA5 Antibody Picoband®
Cat # PB9200
The featured anti-GATA5 antibody is a top-performing, extensively validated reagent for Western blot, with strong literature citation and orthogonal confirmation against negative tissue controls and complementary detection methods, giving confident, reproducible results for reliable GATA5 detection across experimental systems.
Which to pick: Only one Boster GATA5 antibody is catalogued, PB9200, so it is the default choice. It includes an actual Western blot validation image showing specific GATA5 detection under standard SDS-PAGE conditions, supporting its use for your experiment.
Source: BosterBio GATA5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9BWX5.
- Human Protein Atlas. GATA5 tissue expression.