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- Table of Contents
EYA1 / Protein phosphatase EYA1 · Western blot design guide
Design a Western Blot for EYA1
Source-linked EYA1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EYA1 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
EYA1 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~64.6 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 | Ubl conjugation | |
| Caveat | EYA1/4 cross-reactivity | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked EYA1 Western Blot Protocol Options
The A02376 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | A549, H9C2 (catalog A02376) |
| 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 | A02376; 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 EYA1 Western Blot Band Size?
EYA1 has a predicted mass of 64.6 kDa; named isoforms could affect migration, but no empirical band size or distinct isoform bands are established.
| Single band near 64.6 kDa | consistent with the predicted mass of EYA1 |
| Several discrete bands | could reflect EYA1A, EYA1B and EYA1D, if their migration differs |
| One band despite three named isoforms | isoforms may overlap in migration or differ in abundance |
| Stronger band in a nuclear fraction | consistent with the reported nuclear localization of EYA1 |
💡Expected EYA1 appearanceUniProt predicts 64.6 kDa for EYA1; no empirical band size or resolvable isoform pattern is supplied, so confirm any candidate band with ordinary identity controls.
| UniProt predicted mass | places the reference band near 64.6 kDa |
| EYA1A splice isoform | may differ in apparent size from other isoforms; its mass is unspecified |
| EYA1B splice isoform | may differ in apparent size from other isoforms; its mass is unspecified |
| EYA1D splice isoform | may differ in apparent size from other isoforms; its mass is unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | EYA1 abundance in the tested lysate is unknown | check an EYA1 positive control and antibody performance |
| Band higher than expected | identity or isoform migration is unverified | compare with an EYA1 depletion control |
| Band lower than expected | identity or isoform migration is unverified | compare with an EYA1 depletion control |
| Multiple bands | EYA1 isoforms or recognition of EYA4 by the EYA1/4 antibody | use EYA1 depletion and an EYA1 specific antibody to assign bands |
| Weak or no signal | EYA1 may be distributed between cytoplasm and nucleus | check whole cell and nuclear fractions with an EYA1 positive control |
Experiment evidence
Western blot images showing EYA1 band
A02376 · EYA1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EYA1 Western blot
🧪For positive controls for EYA1 in Western blot, you can use a sample with independently confirmed EYA1 expression, since the supplied HPA data identify no positive tissue or cell.
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 such as Ponceau alongside the samples.
⚠️Feasibility: With no HPA expression data, tissue controls cannot be confirmed; a knockdown or KO provides a practical negative control.
HPA tissue expression evidence for EYA1
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 EYA1 Western Blot Tips
Deeper troubleshooting and optimisation questions for EYA1, answered from its protein features.
Where should the EYA1 band appear?
Band shift · The canonical sequence is 592 residues with a predicted mass of 64.6 kDa. Use this as a reference, not a guaranteed apparent band position; no observed band mass is supplied.
Could EYA1 isoforms produce different bands?
Isoforms · Three isoforms are listed: EYA1A, EYA1B and EYA1D. EYA1B replaces canonical residues 1–41 with an eight-residue sequence; EYA1D lacks residues 140–144 and 351–380. These sequence differences may affect band position, but the features do not establish visible separation.
Does EYA1 modification explain a shifted band?
PTM · Ubl conjugation is listed as a keyword, but no modified residue or modification condition is supplied. Do not assign a shifted band to a specific modification from these features alone.
Does this guide establish induction of EYA1?
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.
What transfer method to use for EYA1 Western blot?
Transfer · Choose transfer conditions suitable for a protein near the predicted 64.6 kDa mass, then verify that EYA1 transfers efficiently under your setup. The supplied features do not specify a membrane, transfer mode or observed band position.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02376 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 cytoplasmic and nuclear EYA1 be quantified?
Quantitation · EYA1 is annotated in both cytoplasm and nucleus. Compare like fractions across samples, and keep fraction measurements distinct from total lysate measurements when assessing its localization.
Should DNA damage increase the EYA1 band?
Interpretation · EYA1 localizes to double-strand break sites and interacts with H2AX in response to DNA damage. These features support examining localization after DNA damage; they do not establish an increase in total EYA1 abundance.
Compare them with the predicted 64.6 kDa canonical protein and the listed EYA1B and EYA1D sequence changes. Neither isoform annotations nor the Ubl conjugation keyword identifies an unexpected band by itself; confirm its identity experimentally.
Compare them with the predicted 64.6 kDa canonical protein and the listed EYA1B and EYA1D sequence changes. Neither isoform annotations nor the Ubl conjugation keyword identifies an unexpected band by itself; confirm its identity experimentally.
Boster reagents
EYA1 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-EYA1/4 Antibody
Cat # A02376
The listed Anti-EYA1/4 antibody (A02376) reports human and rat reactivity. Its WB image shows A549 and H9C2 whole cell lysates tested at 1:500 dilution; these examples do not establish performance across other samples or conditions.
Which to pick: A02376 is the only listed option and has a WB image. Consider it for human or rat samples, while checking whether the shown A549 and H9C2 lysate conditions match your experiment.
Source: BosterBio EYA1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q99502.
- Human Protein Atlas. EYA1 tissue expression.
- PMC5739622 — target-verified WB comparison
- PMC13505096 — target-verified WB comparison
- PMC3906160 — target-verified WB comparison