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- Table of Contents
APOA4 / Apolipoprotein A-IV · Western blot design guide
Design a Western Blot for APOA4
Real validated APOA4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-APOA4 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
APOA4 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 | ~45.4 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Duodenum (IHC candidate; verify WB) +2 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Cleaved | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated APOA4 Western Blot Protocols
The A01973 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human testis tissue lysate (catalog A01973) |
| 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 | A01973 · (A) 0.5 and (B) 1 μg/mL (catalog A01973) |
| 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 APOA4 Western Blot Band Size?
APOA4 has a predicted 45.4 kDa precursor; signal-peptide cleavage and homodimerization could affect migration, but no empirical band or demonstrated shift is supplied.
| Band near 45.4 kDa | Consistent with the predicted APOA4 precursor mass; confirm identity with controls. |
| Band below 45.4 kDa | Could reflect removal of the 1..20 signal peptide; its migration is not supplied. |
| Band near twice the monomer mass | Could reflect an APOA4 homodimer if it remains associated during electrophoresis. |
| Little or no band in whole-cell lysate | APOA4 is secreted and may be more abundant outside cells. |
💡Expected APOA4 appearanceUniProt predicts a 45.4 kDa precursor, while signal-peptide cleavage or retained homodimerization could alter migration; no empirical band is supplied, so confirm any candidate band with identity controls.
| Predicted precursor mass | UniProt gives 45.4 kDa as the reference size. |
| Signal peptide at residues 1..20 | Contributes to the full-length precursor mass. |
| Signal-peptide cleavage | Could make mature APOA4 smaller than the precursor; its apparent size is not supplied. |
| Homodimer formation | Could yield a band near twice the monomer mass if the dimer survives electrophoresis. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Secreted APOA4 may be scarce in the cellular fraction. | Check conditioned medium or a suitable extracellular fraction alongside lysate. |
| Band higher than expected | An APOA4 homodimer may remain associated during electrophoresis. | Compare denaturing conditions and verify the band with an independent APOA4 antibody. |
| Band lower than expected | The 1..20 signal peptide may be removed from mature APOA4. | Compare precursor and secreted samples and confirm band identity with an independent antibody. |
| Multiple bands | Precursor and mature APOA4 or retained monomer and homodimer could differ in migration. | Compare cellular and secreted fractions under denaturing conditions and confirm each band’s identity. |
| Weak or no signal | Secreted APOA4 may be underrepresented in the tested sample. | Test an appropriate extracellular sample and include a positive control. |
Experiment evidence
Western blot images showing APOA4 band
A01973 · APOA4
M01973-1 · APOA4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for APOA4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for APOA4 in Western blot, you can use duodenum tissue, which shows high HPA expression.
Positive control: Duodenum (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: Because APOA4 is secreted, whole-cell lysates may give weak signal; consider an extracellular sample.
HPA tissue expression evidence for APOA4
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 |
|---|---|---|---|---|
| Duodenum | endocrine cells | High | Protein (IHC) | HPA → |
| Small intestine | endocrine cells | High | Protein (IHC) | HPA → |
| Kidney | cells in tubules | 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 APOA4 Western Blot Tips
Deeper troubleshooting and optimisation questions for APOA4, answered from its protein features.
How should APOA4 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 annotated APOA4 isoforms explain multiple bands?
Isoforms · UniProt supplies one isoform and no alternative sequence. Its isoform annotation therefore provides no specific explanation for multiple bands. Assess bands using the antibody target and the sample fraction.
What do APOA4 annotations say about modification-dependent bands?
PTM · APOA4 has a phosphoprotein keyword, but no modified residues or glycosylation sites are listed. Do not assign a band to a particular phosphorylation or glycosylation site from these data, or assume a modification produces a visible shift.
Does this guide establish induction of APOA4?
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 APOA4?
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 A01973 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.
Where should APOA4 be measured for quantitative Western blotting?
Quantitation · APOA4 is annotated as secreted, with chylomicron and HDL keywords. Choose and report the sample fraction consistently, and compare equal inputs within that fraction. A cellular lysate alone may not represent the secreted pool.
Why might APOA4 migrate differently from its predicted 45.4 kDa?
Interpretation · The 45.4 kDa prediction includes the precursor. UniProt annotates a signal peptide at residues 1..20, so secreted APOA4 may lack that segment. This feature alone does not establish its apparent band position or explain a measured mass difference; no observed band is supplied.
UniProt places the signal peptide at residues 1..20; the remaining sequence begins at residue 21. Keep precursor and processed protein numbering distinct when comparing antibodies or band assignments. Signal peptide annotation alone does not prove a visible band shift.
UniProt annotates APOA4 as a homodimer, making dimerization a possibility to investigate for an upper band. The annotation does not establish that a dimer persists during Western blot preparation or predict its apparent mass.
UniProt places the signal peptide at residues 1..20; the remaining sequence begins at residue 21. Keep precursor and processed protein numbering distinct when comparing antibodies or band assignments. Signal peptide annotation alone does not prove a visible band shift.
UniProt annotates APOA4 as a homodimer, making dimerization a possibility to investigate for an upper band. The annotation does not establish that a dimer persists during Western blot preparation or predict its apparent mass.
Boster reagents
APOA4 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-Apolipoprotein A-IV ApoA4 Antibody
Cat # A01973
Real WB data
Anti-ApoA4 Rabbit Monoclonal Antibody
Cat # M01973-1
The catalog reports two anti-APOA4 antibodies with Western blot images: A01973 in human testis lysate at 0.5 and 1 μg/mL, and M01973-1 in human plasma lysate. These examples show specific tested samples; broader Western blot performance is not established here.
Which to pick: For human testis lysate, consider A01973; for human plasma lysate, consider M01973-1. A01973 also lists chicken reactivity, but the supplied Western blot caption documents a human sample only.
Source: BosterBio APOA4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P06727.
- Human Protein Atlas. APOA4 tissue expression.
- PMC12059330 — target-verified WB comparison
- PMC8225859 — target-verified WB comparison
- PMC8123842 — target-verified WB comparison