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- Table of Contents
ASCL1 / Achaete-scute homolog 1 · Western blot design guide
Design a Western Blot for ASCL1
Real validated ASCL1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ASCL1 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
ASCL1 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 | ~25.5 kDa | |
| Observed band | ~30 kDa | |
| Gel | 5–20% (catalog A03023-2) | |
| Positive control | Cerebellum (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 | Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated ASCL1 Western Blot Protocols
The A03023-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human K562, human U251, human PC-3 (catalog A03023-2) |
| Gel % | 5–20% (catalog A03023-2) |
| Load | 30 ug; reducing conditions (catalog A03023-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A03023-2) |
| Membrane | nitrocellulose membrane (catalog A03023-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A03023-2) |
| Primary antibody | A03023-2 · 0.5 μg/mL (catalog A03023-2) |
| Primary incubation | overnight at 4°C (catalog A03023-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A03023-2) |
| Secondary incubation | 1.5 hour at RT (catalog A03023-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A03023-2) |
| Detection | ECL (catalog A03023-2) |
Section 2
What Is the Expected ASCL1 Western Blot Band Size?
ASCL1 is predicted at 25.5 kDa and observed near 30 kDa; the cause of the difference is not established.
| Band near 30 kDa | Matches the empirical ASCL1 band; confirm identity with a specificity control. |
| Band near 25.5 kDa | Matches the predicted mass alone; its identity requires confirmation. |
| Faint band in whole-cell lysate | Nuclear ASCL1 may be diluted in the sample. |
| Stronger band in a nuclear fraction | Consistent with ASCL1's nuclear location; confirm band identity. |
💡Expected ASCL1 appearanceASCL1 has a predicted mass of 25.5 kDa, while antibody QC shows a band near 30 kDa; the cause of the difference is unestablished, so confirm identity with ordinary controls.
| Predicted molecular mass | Sets a 25.5 kDa reference for full-length ASCL1. |
| Full-length mass reference | Does not establish where ASCL1 migrates on a blot. |
| Predicted versus empirical mass | The empirical band appears near 30 kDa, above the 25.5 kDa prediction. |
| Calculated mass in daltons | The supplied 25,454 Da value corresponds to approximately 25.5 kDa. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear ASCL1 may be poorly represented in the sampled lysate. | Check a nuclear fraction and verify sample loading. |
| Band higher than expected | The observed band is near 30 kDa, above the 25.5 kDa prediction; the cause is unestablished. | Compare with antibody QC and confirm identity with a specificity control. |
| Band lower than expected | A lower band's identity is not established by the supplied evidence. | Check sample integrity and use a specificity control. |
| Multiple bands | Additional bands are not explained by the single listed isoform. | Confirm each band's identity with a specificity control. |
| Weak or no signal | The sample may contain little detectable nuclear ASCL1. | Check nuclear enrichment, loading, and antibody performance. |
| Fragments below expected size | Sample degradation is possible; no ASCL1 cleavage product is listed. | Assess sample integrity and confirm fragment identity. |
Experiment evidence
Western blot images showing ASCL1 band
A03023-2 · ASCL1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ASCL1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ASCL1 in Western blot, you can use cerebellum tissue, which shows high expression in HPA.
Positive control: Cerebellum (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: ASCL1 is nuclear, so a nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for ASCL1
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 |
|---|---|---|---|---|
| Cerebellum | processes in granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | High | Protein (IHC) | HPA → |
| Hippocampus | glial cells | High | Protein (IHC) | HPA → |
| Caudate | glial 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 | endocrine 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 ASCL1 Western Blot Tips
Deeper troubleshooting and optimisation questions for ASCL1, answered from its protein features.
How should ASCL1 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.
Do the supplied features support multiple ASCL1 isoform bands?
Isoforms · Only one isoform is listed, with no alternative sequence supplied. Do not assign additional bands to splice isoforms from these features alone; verify any additional signal independently.
Could acetylation shift the ASCL1 band?
PTM · UniProt lists N6-acetyllysine at position 156 in the supplied sequence. This establishes a modification site, but does not show that acetylation causes a visible shift. Keep the UniProt coordinate when comparing reports or antibody information that may use different numbering.
Does this guide establish induction of ASCL1?
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 ASCL1?
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 A03023-2 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.
Which band should be used to quantify ASCL1?
Quantitation · The supplied observed band is approximately 30 kDa. Verify that the band being measured represents ASCL1, then quantify the same band consistently across samples. The predicted 25.5 kDa mass alone does not establish band identity.
Why might ASCL1 appear near 30 kDa instead of 25.5 kDa?
Interpretation · The supplied observed band is approximately 30 kDa, while the predicted mass is 25.5 kDa. The listed features do not establish why they differ. Use the approximately 30 kDa band as a candidate and verify its identity before interpreting the difference.
ASCL1 is annotated as nuclear. A nuclear fraction is therefore relevant when assessing its signal, and fraction choice should be consistent across samples being compared.
Efficient DNA binding requires dimerization with another bHLH protein, and ASCL1 forms a heterodimer with TCF3. That interaction alone does not identify a higher Western blot band as an ASCL1–TCF3 complex.
Compare them with the approximately 30 kDa observed band and verify their identity. The supplied features list one isoform and acetylation at UniProt position 156, but neither feature alone assigns an unexpected band or demonstrates a visible shift.
ASCL1 is annotated as nuclear. A nuclear fraction is therefore relevant when assessing its signal, and fraction choice should be consistent across samples being compared.
Efficient DNA binding requires dimerization with another bHLH protein, and ASCL1 forms a heterodimer with TCF3. That interaction alone does not identify a higher Western blot band as an ASCL1–TCF3 complex.
Compare them with the approximately 30 kDa observed band and verify their identity. The supplied features list one isoform and acetylation at UniProt position 156, but neither feature alone assigns an unexpected band or demonstrates a visible shift.
Boster reagents
ASCL1 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-ASCL1 Antibody Picoband®
Cat # A03023-2
The listed anti-ASCL1 antibody, A03023-2, is a rabbit polyclonal with reported human, mouse, and rat reactivity. Its Western blot image shows a band near 30 kDa, versus an expected 25 kDa; the supplied evidence is a product image, with no publication evidence provided.
Which to pick: A03023-2 is the only listed option and has a Western blot image using human cell, rat brain and cell, and mouse brain and cell lysates. Check whether your sample resembles those tested contexts; the caption does not establish performance in every tissue or cell type.
Source: BosterBio ASCL1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P50553.
- Human Protein Atlas. ASCL1 tissue expression.
- PMC11903614 — target-verified WB comparison
- PMC11830715 — target-verified WB comparison
- PMC11217739 — target-verified WB comparison