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HEY1 / Hairy/enhancer-of-split related with YRPW motif protein 1 · Western blot design guide
Design a Western Blot for HEY1
Real validated HEY1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HEY1 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
HEY1 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 | ~32.6 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Cerebral cortex (IHC candidate; verify WB) +3 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | — | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated HEY1 Western Blot Protocols
The A02454-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of various cell lines, (catalog A02454-1) |
| 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 | Blocking buffer: 3% nonfat dry milk in TBST (catalog A02454-1) |
| Primary antibody | A02454-1 · 1:1000 (catalog A02454-1) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A02454-1) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A02454-1) |
Section 2
What Is the Expected HEY1 Western Blot Band Size?
HEY1 is predicted at 32.6 kDa; isoforms and self-association could affect patterns, but altered migration has not been demonstrated.
| Band near 32.6 kDa | consistent with the predicted HEY1 mass; identity requires controls |
| Single sharp band | isoforms 1 and 2 need not resolve as separate bands |
| Multiple discrete bands | may reflect isoforms 1 and 2; their migration difference is unknown |
| Band enriched in nuclear extract | consistent with HEY1 nuclear localization |
| Higher-mass species | could reflect self-association if complexes survive preparation; size is unknown |
💡Expected HEY1 appearanceHEY1 has a predicted mass of 32.6 kDa, but no empirical band size is supplied; use band-identity controls to assess any observed band.
| Predicted HEY1 mass | 32.6 kDa is the sequence-based reference size, not validated migration |
| Splice isoform 1 | its individual mass and migration are not supplied |
| Splice isoform 2 | may differ from isoform 1, but its mass and migration are not supplied |
| Self-association | could produce a higher-mass species if complexes persist; no oligomer size is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | HEY1 is nuclear; its abundance in the sampled lysate is unknown | check a nuclear extract and a suitable positive control |
| Band higher than expected | self-association is possible, but the band identity is unverified | compare prepared samples and confirm the band with an independent antibody or HEY1 depletion |
| Band lower than expected | isoform migration or fragmentation is possible; neither size is documented | check antibody epitope coverage and confirm with HEY1 depletion |
| Multiple bands | isoforms 1 and 2 exist, but distinct migration is unproven | compare isoform-aware controls and assess specificity with HEY1 depletion |
| Weak or no signal | nuclear HEY1 may be poorly represented in the sampled fraction | test nuclear enrichment alongside a positive control |
| Fragments below expected size | fragment identity is not established by the supplied features | check sample integrity and confirm HEY1 dependence |
Experiment evidence
Western blot images showing HEY1 band
A02454-1 · HEY1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HEY1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for HEY1 in Western blot, you can use cerebral cortex tissue, which HPA scores High.
Positive control: Cerebral cortex (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: HEY1 is nuclear, so nuclear enrichment may help produce a clear signal.
HPA tissue expression evidence for HEY1
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 |
|---|---|---|---|---|
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | Medium | Protein (IHC) | HPA → |
| Hippocampus | 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 → |
| 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 HEY1 Western Blot Tips
Deeper troubleshooting and optimisation questions for HEY1, answered from its protein features.
What band size should I expect for HEY1?
Band shift · The canonical HEY1 sequence is 304 amino acids with a predicted mass of 32.6 kDa. No observed Western blot band size is supplied, so use 32.6 kDa as a reference, not a guaranteed apparent position.
Could HEY1 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. At canonical UniProt position 82, isoform 2 replaces K with KQVME, adding four residues. The sequence difference may affect apparent mobility, but the supplied features do not establish that two bands will resolve.
Do listed modifications explain a shifted HEY1 band?
PTM · No modified residues or glycosylation sites are listed. The supplied features therefore do not identify a modification that explains a shift. They also do not establish why an observed band might differ from the calculated mass.
Does this guide establish induction of HEY1?
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 HEY1?
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 A02454-1 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.
How should HEY1 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.
Which sample fraction is relevant for detecting HEY1?
Interpretation · HEY1 is annotated as nuclear. A nuclear fraction is therefore a relevant sample to examine, especially if a whole-cell sample gives a weak signal. The annotation does not specify an extraction protocol or guarantee stronger detection.
Use the same sample type and band assignment across comparisons. HEY1 is nuclear and has two listed isoforms, so a change in nuclear recovery or in which isoform a band represents could complicate interpretation. The supplied features do not identify a preferred normalization control.
Check each band's position against the 32.6 kDa predicted mass and consider the two listed isoforms. HEY1 self-associates and interacts with other proteins, but those features alone do not establish that an extra Western blot band is a complex. No empirical HEY1 band pattern is supplied.
Use the same sample type and band assignment across comparisons. HEY1 is nuclear and has two listed isoforms, so a change in nuclear recovery or in which isoform a band represents could complicate interpretation. The supplied features do not identify a preferred normalization control.
Check each band's position against the 32.6 kDa predicted mass and consider the two listed isoforms. HEY1 self-associates and interacts with other proteins, but those features alone do not establish that an extra Western blot band is a complex. No empirical HEY1 band pattern is supplied.
Boster reagents
HEY1 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-HEY1 Antibody
Cat # A02454-1
A02454-1 is an anti-HEY1 antibody listed for Western blotting with human, mouse, and rat reactivity. Its WB image uses extracts from various cell lines at 25 µg per lane and a 1:1000 primary dilution; the caption does not identify the cell lines.
Which to pick: A02454-1 is the only listed option and has a WB image. Use its reported 1:1000 primary dilution as a starting point, and confirm performance in your specific sample.
Source: BosterBio HEY1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9Y5J3.
- Human Protein Atlas. HEY1 tissue expression.
- PMC7864008 — target-verified WB comparison
- PMC5546488 — target-verified WB comparison
- PMC4385965 — target-verified WB comparison