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- Table of Contents
FOXO4 / Forkhead box protein O4 · Western blot design guide
Design a Western Blot for FOXO4
Real validated FOXO4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-FOXO4 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
FOXO4 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 | ~53.7 kDa | |
| Observed band | ~65 kDa | |
| Gel | 5–20% (catalog M01819-1) | |
| Positive control | Heart muscle (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated FOXO4 Western Blot Protocols
The M01819-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human K562, human Hela, human 293T (catalog M01819-1) |
| Gel % | 5–20% (catalog M01819-1) |
| Load | 30 ug; reducing conditions (catalog M01819-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M01819-1) |
| Membrane | nitrocellulose membrane (catalog M01819-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M01819-1) |
| Primary antibody | M01819-1 · 1:500 (catalog M01819-1) |
| Primary incubation | overnight at 4°C (catalog M01819-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:500 (catalog M01819-1) |
| Secondary incubation | 1.5 hour at RT (catalog M01819-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M01819-1) |
| Detection | ECL (catalog M01819-1) |
Section 2
What Is the Expected FOXO4 Western Blot Band Size?
FOXO4 is predicted at 53.7 kDa and observed near 65 kDa; the cause of the difference is not established.
| Band near 65 kDa | Empirical FOXO4 band; confirm identity with appropriate controls |
| Band near 53.7 kDa | Near the predicted FOXO4 mass; identity requires confirmation |
| Additional discrete bands | Isoforms 1 and Zeta are possible, but their migration is not established |
| Weak nuclear band with cytoplasmic signal | Phosphorylated FOXO4 can redistribute from nucleus to cytoplasm |
💡Expected FOXO4 appearanceFOXO4 has a predicted mass of 53.7 kDa and an empirical band near 65 kDa; the cause of the difference is unestablished, so confirm band identity with appropriate controls.
| Predicted FOXO4 mass | 53.7 kDa calculated; the empirical band is near 65 kDa |
| Isoforms 1 and Zeta | May differ in size; their masses and band separation are not supplied |
| PKB/AKT1 phosphorylation at Thr32 | May affect migration; no visible shift is established |
| PKB/AKT1 phosphorylation at Ser197 | May affect migration; no visible shift is established |
| PKB/AKT1 phosphorylation at Ser262 | May affect migration; no visible shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | FOXO4 signal may be below detection | Check loading and detection with a positive control lysate |
| Band higher than expected | The empirical FOXO4 band is near 65 kDa despite a 53.7 kDa prediction; the cause is unestablished | Compare with the reported 65 kDa band and verify identity using an independent antibody or FOXO4 depletion |
| Band lower than expected | Band identity or an isoform difference is uncertain | Verify identity with FOXO4 depletion and assess isoform expression |
| Multiple bands | FOXO4 has isoforms 1 and Zeta, but their separation is unestablished | Use FOXO4 depletion to identify specific bands |
| Weak or no signal | FOXO4 redistributes between nucleus and cytoplasm | Check both fractions and a positive control lysate |
Experiment evidence
Western blot images showing FOXO4 band
M01819-1 · FOXO4
A01819 · FOXO4
M01819 · FOXO4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for FOXO4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for FOXO4 in Western blot, you can use heart muscle lysate.
Positive control: Heart muscle (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 samples.
⚠️Feasibility: FOXO4 shifts between nucleus and cytoplasm, so signal in subcellular fractions may vary.
HPA tissue expression evidence for FOXO4
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 |
|---|---|---|---|---|
| Heart muscle | cardiomyocytes | Medium | Protein (IHC) | HPA → |
| Placenta | cytotrophoblasts | Medium | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Medium | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Medium | Protein (IHC) | HPA → |
| Testis | Leydig 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 FOXO4 Western Blot Tips
Deeper troubleshooting and optimisation questions for FOXO4, answered from its protein features.
How should FOXO4 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 FOXO4 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and Zeta. Zeta lacks residues 58–112 of the canonical sequence, so the isoforms may migrate differently. Compare isoform-specific expression or detection before assigning multiple bands; the sequence difference alone does not establish their apparent sizes.
Which FOXO4 phosphorylation sites are relevant to a blot?
PTM · PKB/AKT1 phosphorylates Thr32, Ser197 and Ser262 in the supplied UniProt coordinates. If testing phosphorylation, use site-specific detection or a phosphorylation-sensitive comparison alongside total FOXO4. Check the numbering convention of any antibody before matching its stated site to these coordinates.
The supplied localization annotation says phosphorylation moves FOXO4 from nucleus to cytoplasm, while dephosphorylation promotes nuclear translocation. Measure both fractions when interpreting a treatment-related change; a decrease in one fraction need not mean a decrease in total FOXO4.
The supplied annotations report acetylation by CREBBP/CBP, deacetylation by SIRT1, and changes in localization with monoubiquitination and deubiquitination. These features motivate checking localization or modification-specific signals when bands change, but do not by themselves predict a visible shift.
The supplied localization annotation says phosphorylation moves FOXO4 from nucleus to cytoplasm, while dephosphorylation promotes nuclear translocation. Measure both fractions when interpreting a treatment-related change; a decrease in one fraction need not mean a decrease in total FOXO4.
The supplied annotations report acetylation by CREBBP/CBP, deacetylation by SIRT1, and changes in localization with monoubiquitination and deubiquitination. These features motivate checking localization or modification-specific signals when bands change, but do not by themselves predict a visible shift.
Does this guide establish induction of FOXO4?
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 FOXO4?
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 M01819-1 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 FOXO4 be quantified across conditions?
Quantitation · Quantify a verified FOXO4 band consistently across samples. Because FOXO4 can move between nucleus and cytoplasm, compare like fractions and consider whole-cell measurements when assessing total abundance. Distinguish total FOXO4 from phosphorylation-specific signal.
Why might FOXO4 appear near 65 kDa instead of 53.7 kDa?
Interpretation · The predicted mass is 53.7 kDa, while the reported apparent band is about 65 kDa. FOXO4 has phosphorylation and other reported modifications, but these features alone do not establish the cause of the difference. Use the observed band as a reference and verify its identity experimentally.
Consider the Zeta isoform, which lacks canonical residues 58–112, and the reported modifications as possibilities. Neither feature alone identifies an extra band. Confirm band identity with an independent antibody or FOXO4 depletion, and avoid assigning a specific modification from apparent size alone.
Consider the Zeta isoform, which lacks canonical residues 58–112, and the reported modifications as possibilities. Neither feature alone identifies an extra band. Confirm band identity with an independent antibody or FOXO4 depletion, and avoid assigning a specific modification from apparent size alone.
Boster reagents
FOXO4 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-FoxO4/Afx Rabbit Monoclonal Antibody
Cat # M01819-1
Real WB data
Anti-Forkhead box protein O4 FOXO4 Antibody
Cat # A01819
Real WB data
Anti-FoxO4/Afx Rabbit Monoclonal Antibody
Cat # M01819
All three anti-FOXO4 antibodies have WB images. M01819-1 shows human cell, rat skeletal muscle, and mouse heart lysates, with a band near 65 kDa versus 54 kDa expected. A01819 shows HeLa lysate; M01819 shows FOXO4-transfected 293T lysate.
Which to pick: For the widest range of pictured samples, consider M01819-1. A01819 has a HeLa WB image; M01819 has a transfected 293T WB image and lists human reactivity only. Match your sample to the reported context and check the band-size discrepancy for M01819-1.
Source: BosterBio FOXO4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P98177.
- Human Protein Atlas. FOXO4 tissue expression.
- PMC8256341 — target-verified WB comparison
- PMC4070918 — target-verified WB comparison
- PMC8149257 — target-verified WB comparison