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- Table of Contents
APAF1 / Apoptotic protease-activating factor 1 · Western blot design guide
Design a Western Blot for APAF1
Real validated APAF1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-APAF1 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
APAF1 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 | ~141.8 kDa | |
| Observed band | ~135 kDa | |
| Gel | 5–20% (catalog A00889-2) | |
| Positive control | Bone marrow (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 | — | |
| Caveat | Observed mass differs | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 6 isoform(s) |
Section 1
Real Curated APAF1 Western Blot Protocols
The A00889-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HEL, human HL-60 (catalog A00889-2) |
| Gel % | 5–20% (catalog A00889-2) |
| Load | 30 ug; reducing conditions (catalog A00889-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00889-2) |
| Membrane | nitrocellulose membrane (catalog A00889-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00889-2) |
| Primary antibody | A00889-2 · 0.5 μg/mL (catalog A00889-2) |
| Primary incubation | overnight at 4°C (catalog A00889-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00889-2) |
| Secondary incubation | 1.5 hour at RT (catalog A00889-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00889-2) |
| Detection | ECL (catalog A00889-2) |
Section 2
What Is the Expected APAF1 Western Blot Band Size?
APAF1 predicts 141.8 kDa and shows an empirical band near 135 kDa; the cause of the difference is not established.
| Band near 135 kDa | Empirical APAF1 band in whole-cell lysate under reducing conditions |
| Band near 141.8 kDa | Near the UniProt predicted mass; confirm its identity |
| Additional bands at different masses | Could reflect APAF1 isoforms 1–6, whose migration is unreported |
| No detectable band in lysate | Cytoplasmic APAF1 may be below detection in this sample |
💡Expected APAF1 appearanceUniProt predicts 141.8 kDa, while antibody QC detects an APAF1 band near 135 kDa in reducing whole-cell lysates; the reason for the difference is unestablished, so confirm band identity with appropriate controls.
| UniProt predicted mass | Full-length sequence predicts 141.8 kDa; the empirical band is near 135 kDa |
| Splice isoforms 1 and 2 | Their individual masses and relative migration are not supplied |
| Splice isoforms 3 and 4 | Their individual masses and relative migration are not supplied |
| Splice isoforms 5 and 6 | Their individual masses and relative migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Cytoplasmic APAF1 may be below detection in the tested sample | Check lysate quality and compare with an APAF1-positive lysate |
| Band higher than expected | The higher band's identity and cause are unestablished | Compare with the 135 kDa QC band and verify specificity by APAF1 knockdown |
| Band lower than expected | An isoform, proteolysis, or nonspecific binding may account for the band | Compare antibodies recognizing different epitopes and verify by APAF1 knockdown |
| Multiple bands | Isoforms 1–6 are annotated, but distinct band sizes are unreported | Check each band's response to APAF1 knockdown |
| Weak or no signal | APAF1 may be below the assay's detection limit in this lysate | Check loading and antibody performance with a positive lysate |
| Fragments below expected size | Proteolysis or nonspecific binding may produce smaller bands | Use protease inhibitors during preparation and verify specificity by APAF1 knockdown |
Experiment evidence
Western blot images showing APAF1 band
A00889-2 · APAF1
M00889 · APAF1
A00889 · APAF1
M00889-2 · APAF1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for APAF1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for APAF1 in Western blot, you can use bone marrow lysate, the highest-scoring HPA tissue.
Positive control: Bone marrow (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: Cytoplasmic lysates are suitable, but verify the expected lack of signal in adipose tissue.
HPA tissue expression evidence for APAF1
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 |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Colon | endothelial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Medium | Protein (IHC) | HPA → |
| Endometrium | glandular 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 → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced APAF1 Western Blot Tips
Deeper troubleshooting and optimisation questions for APAF1, answered from its protein features.
How should APAF1 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.
Which APAF1 isoforms could yield different bands?
Isoforms · UniProt lists six isoforms. Relative to the canonical sequence, isoforms 2 and 3 lack residues 99–109; isoforms 3, 4, and 5 lack 824–866. Isoform 5 also has changes at 575 and lacks 1113–1154. Isoform 6 changes 319–338 and lacks 339–1248, so it could be substantially smaller. These are UniProt coordinates.
Check the antibody’s mapped epitope against the UniProt alternative-sequence regions. An epitope within residues 339–1248 cannot detect isoform 6; one within 824–866 cannot detect isoforms 3, 4, or 5. Use UniProt coordinates when comparing regions, since antibody documentation may use different numbering.
Check the antibody’s mapped epitope against the UniProt alternative-sequence regions. An epitope within residues 339–1248 cannot detect isoform 6; one within 824–866 cannot detect isoforms 3, 4, or 5. Use UniProt coordinates when comparing regions, since antibody documentation may use different numbering.
Do annotated APAF1 modifications explain a shifted band?
PTM · The supplied UniProt features list no modified residues or glycosylation sites. They therefore provide no specific modification to assign to a band shift. A change in apparent mass alone does not identify a modification.
Does this guide establish induction of APAF1?
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.
What transfer method to use for APAF1 Western blot?
Transfer · Choose transfer conditions suitable for a protein around the canonical predicted mass of 141.8 kDa. Check signal on the membrane and residual protein in the gel to assess transfer. The supplied features do not specify a membrane type, transfer time, or current.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00889-2 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.
How should APAF1 bands be quantified?
Quantitation · Use exposures within the signal’s linear range and a consistent loading reference. Decide whether to quantify one band or the sum of detected bands based on the isoforms recognized by the antibody. APAF1 is annotated as cytoplasmic, so keep the sample fraction consistent across comparisons.
Why might APAF1 appear near 135 kDa instead of 141.8 kDa?
Interpretation · 141.8 kDa is the predicted mass, while ~135 kDa is an observed apparent band. These values need not match exactly. The supplied features do not establish what causes the difference; compare the band with an appropriate molecular-weight marker and consider which isoforms the antibody detects.
No abundance change is specified. UniProt describes cytoplasmic APAF1 as a monomer that oligomerizes into a heptameric apoptosome upon binding cytochrome c and dATP. Quantify APAF1 bands directly if testing abundance; a band’s intensity alone does not establish apoptosome formation.
First compare their sizes with the six annotated isoforms and check whether the antibody epitope is retained. Isoform 6 lacks UniProt residues 339–1248 and may yield a much smaller product. The features alone cannot establish the identity of an unexpected band or show that oligomerization or a modification caused it.
No abundance change is specified. UniProt describes cytoplasmic APAF1 as a monomer that oligomerizes into a heptameric apoptosome upon binding cytochrome c and dATP. Quantify APAF1 bands directly if testing abundance; a band’s intensity alone does not establish apoptosome formation.
First compare their sizes with the six annotated isoforms and check whether the antibody epitope is retained. Isoform 6 lacks UniProt residues 339–1248 and may yield a much smaller product. The features alone cannot establish the identity of an unexpected band or show that oligomerization or a modification caused it.
Boster reagents
APAF1 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-APAF1 Antibody Picoband®
Cat # A00889-2
Real WB data
Anti-APAF1 Rabbit Monoclonal Antibody
Cat # M00889
Real WB data
Anti-Apaf1 Antibody
Cat # A00889
Real WB data
Anti-Apaf-1 Monoclonal Antibody [2E10]
Cat # M00889-2
Four the supplier anti-APAF1 antibodies have WB images. Captions show human cell lysates, including HEL, HL-60, HeLa, MCF-7, and K562. A00889-2 shows a band near the expected 135 kDa; the other captions provide less detail. Mouse and rat WB examples are not supplied.
Which to pick: For human samples, A00889-2 has the most detailed WB caption. M00889 also lists mouse reactivity; A00889 and M00889-2 list both mouse and rat reactivity. All four have WB images, but the supplied captions show only human cell lines.
Source: BosterBio APAF1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O14727.
- Human Protein Atlas. APAF1 tissue expression.
- PMC2198828 — target-verified WB comparison
- PMC6599840 — target-verified WB comparison
- PMC11914182 — target-verified WB comparison