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- Table of Contents
NPAS4 / Neuronal PAS domain-containing protein 4 · Western blot design guide
Design a Western Blot for NPAS4
Source-linked NPAS4 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NPAS4 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
NPAS4 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 | ~87.1 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Caudate (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 | Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked NPAS4 Western Blot Protocol Options
The A02045 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HEK293T, RAW264.7 (catalog A02045) |
| Gel % | 8–10% (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 | A02045; 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 NPAS4 Western Blot Band Size?
NPAS4 has a predicted mass of 87.1 kDa; isoforms could affect band size, but their migration has not been demonstrated here.
| Band near 87.1 kDa | Consistent with the predicted NPAS4 mass; identity requires confirmation |
| Single band near 87.1 kDa | Compatible with NPAS4 even though two isoforms are listed |
| Several bands at different positions | Isoforms 1 and 2 are possible contributors, but their migration is unknown |
| Stronger band in nuclear fraction than whole-cell lysate | Consistent with NPAS4 nuclear localization |
💡Expected NPAS4 appearanceUniProt predicts NPAS4 at 87.1 kDa; no empirical band size is supplied, and isoform migration is unknown, so confirm band identity with appropriate controls.
| Predicted molecular weight | Places the reference size at 87.1 kDa |
| Alternative splicing | Produces isoforms 1 and 2, whose size difference is unspecified |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | Its individual mass and migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear NPAS4 may be poorly recovered in the lysate | Check nuclear extraction and compare a nuclear fraction |
| Band higher than expected | The supplied features do not establish an upward shift | Confirm identity by NPAS4 knockdown or an independent antibody |
| Band lower than expected | An alternative isoform is possible, but its size is unknown | Confirm identity by NPAS4 knockdown or an independent antibody |
| Multiple bands | Isoforms 1 and 2 are listed, but distinct migration is unconfirmed | Test band identity with isoform-specific reagents if available |
| Weak or no signal | Nuclear localization may limit recovery from whole-cell lysate | Check nuclear recovery and compare a nuclear fraction |
Experiment evidence
Western blot images showing NPAS4 band
A02045 · NPAS4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NPAS4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NPAS4 in Western blot, you can use caudate tissue, which HPA reports as positive.
Positive control: Caudate (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: NPAS4 is nuclear, so nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for NPAS4
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 |
|---|---|---|---|---|
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | 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 → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NPAS4 Western Blot Tips
Deeper troubleshooting and optimisation questions for NPAS4, answered from its protein features.
How should NPAS4 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 NPAS4 isoforms produce different bands?
Isoforms · Yes. UniProt lists two isoforms. Relative to canonical isoform 1, isoform 2 has V234G and lacks residues 235–802 in UniProt numbering. It is substantially shorter, but the features do not establish where either isoform migrates on a blot.
Choose an epitope within residues 1–233 of the canonical sequence if detection of both listed isoforms is the goal. An epitope within residues 235–802 is absent from isoform 2. Confirm the antibody's stated epitope against UniProt numbering.
Decide whether the measurement targets isoform 1, isoform 2, or both. Isoform 2 lacks canonical residues 235–802, so antibody epitope coverage matters. Quantify a consistently identified band across samples; the supplied features give no observed band position.
Choose an epitope within residues 1–233 of the canonical sequence if detection of both listed isoforms is the goal. An epitope within residues 235–802 is absent from isoform 2. Confirm the antibody's stated epitope against UniProt numbering.
Decide whether the measurement targets isoform 1, isoform 2, or both. Isoform 2 lacks canonical residues 235–802, so antibody epitope coverage matters. Quantify a consistently identified band across samples; the supplied features give no observed band position.
Can a modification explain an unexpected NPAS4 band shift?
PTM · UniProt lists Ubl conjugation as a keyword, but supplies no modified residue or conjugation site. That annotation alone cannot establish a visible shift or explain an observed band. Do not assign an unexpected band to a specific modification from these features.
Does this guide establish induction of NPAS4?
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 NPAS4?
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 A02045 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 NPAS4 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.
Should NPAS4 migrate at its predicted 87.1 kDa?
Interpretation · 87.1 kDa is the predicted mass of the 802-residue canonical protein. No observed Western blot band is supplied, so these features cannot establish its apparent migration or explain a difference.
UniProt places NPAS4 in the nucleus. A nuclear fraction is therefore relevant when assessing its detection; compare the same fraction across samples when quantifying a band.
NPAS4 forms heterodimers with ARNT, ARNT2, or BMAL1 for efficient DNA binding. This interaction does not establish that a dimer survives Western blot preparation or produces a high band. Verify an unexpected band's identity before assigning it to a complex.
UniProt places NPAS4 in the nucleus. A nuclear fraction is therefore relevant when assessing its detection; compare the same fraction across samples when quantifying a band.
NPAS4 forms heterodimers with ARNT, ARNT2, or BMAL1 for efficient DNA binding. This interaction does not establish that a dimer survives Western blot preparation or produces a high band. Verify an unexpected band's identity before assigning it to a complex.
Boster reagents
NPAS4 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-NPAS4 Antibody
Cat # A02045
A02045 is an anti-NPAS4 polyclonal antibody listed as reactive with human and mouse. Its WB image shows testing at 1:500 with HEK293T and RAW264.7 whole-cell lysates. No publication evidence is supplied.
Which to pick: A02045 is the only listed option. Its WB image documents HEK293T and RAW264.7 whole-cell lysates at 1:500; use those tested contexts when assessing fit for your samples.
Source: BosterBio NPAS4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q8IUM7.
- Human Protein Atlas. NPAS4 tissue expression.
- PMC3478397 — target-verified WB comparison
- PMC11089401 — target-verified WB comparison
- PMC6065191 — target-verified WB comparison