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AFAP1L1 / Actin filament-associated protein 1-like 1 · Western blot design guide
Design a Western Blot for AFAP1L1
Source-linked AFAP1L1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-AFAP1L1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled October 2026 · For research use; verify linked source records and product datasheet before use
AFAP1L1 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 | ~86.4 kDa | |
| Observed band | 68 kDa | |
| Gel | 8–10% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 4 isoform(s) |
Section 1
Source-Linked AFAP1L1 Western Blot Protocol Options
The A11242 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | A549 cell lysate (catalog A11242) |
| 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 | A11242 · 1 μg/mL (catalog A11242) |
| 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 AFAP1L1 Western Blot Band Size?
AFAP1L1 is predicted at 86.4 kDa, while antibody QC shows 68 kDa in A549 lysate; the cause of the difference is not established.
| Band at 68 kDa in A549 lysate | Empirical AFAP1L1 antibody signal; confirm identity with knockdown or a second antibody |
| Band near 86.4 kDa | Compatible with the predicted mass of full-length AFAP1L1 |
| Several bands at different sizes | Could reflect isoforms 1, 2, 3, and 4; their migration is unestablished |
| Band in a cytoplasmic fraction | Consistent with AFAP1L1 localization in the cytoplasm and cytoskeleton |
💡Expected AFAP1L1 appearanceUniProt predicts 86.4 kDa, while antibody QC reports a 68 kDa band in A549 lysate; confirm its identity by knockdown or a second antibody before assigning the difference.
| Predicted full-length mass | 86.4 kDa by sequence; the observed antibody signal is 68 kDa, with no established cause for the difference |
| Splice isoform 1 | Its individual apparent size is not supplied |
| Splice isoform 2 | Its size relative to isoform 1 is not established |
| Splice isoform 3 | Its size relative to the other isoforms is not established |
| Splice isoform 4 | Its size relative to the other isoforms is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Signal may be weak in the sampled lysate despite cytoplasmic and cytoskeletal localization | Compare with A549 lysate and check cytoplasmic or cytoskeletal fractions |
| Band higher than expected | The identity of an additional higher band is unestablished | Compare reducing conditions and test signal loss after AFAP1L1 knockdown |
| Band lower than expected | The reported 68 kDa signal is below the 86.4 kDa sequence prediction; its cause is unknown | Confirm the 68 kDa band by AFAP1L1 knockdown and a second antibody |
| Multiple bands | Four splice isoforms are listed, but their migration is unknown | Check which bands decrease after AFAP1L1 knockdown |
| Fragments below expected size | Smaller signals could be degradation products or unrelated bands | Use fresh lysate with protease inhibitors and confirm band identity by knockdown |
Experiment evidence
Western blot images showing AFAP1L1 band
A11242 · AFAP1L1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for AFAP1L1 Western blot
🧪For positive controls for AFAP1L1 in Western blot, you can use no HPA-supported tissue or cell line because no positive sample was supplied.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: With no HPA tissue data, a positive sample needs empirical validation and a knockdown or KO line can provide a negative control.
HPA tissue expression evidence for AFAP1L1
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced AFAP1L1 Western Blot Tips
Deeper troubleshooting and optimisation questions for AFAP1L1, answered from its protein features.
How should AFAP1L1 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 AFAP1L1 isoforms could produce different bands?
Isoforms · UniProt lists isoforms 1–4. Relative to the canonical sequence, isoform 2 lacks residues 719–761; isoform 3 replaces residues 341–377 and lacks 378–768; isoform 4 lacks 1–385. These sequence differences could affect band size and antibody recognition. They do not establish the identity of any observed band.
Compare the mapped epitope with the UniProt alternative sequences. An epitope within residues 719–761 is absent from isoform 2; one within 378–768 is absent from isoform 3; one within 1–385 is absent from isoform 4. Isoform 3 also replaces residues 341–377, which may affect recognition there. Confirm the antibody’s stated epitope and numbering convention.
Compare the mapped epitope with the UniProt alternative sequences. An epitope within residues 719–761 is absent from isoform 2; one within 378–768 is absent from isoform 3; one within 1–385 is absent from isoform 4. Isoform 3 also replaces residues 341–377, which may affect recognition there. Confirm the antibody’s stated epitope and numbering convention.
Which phosphorylation sites matter when interpreting AFAP1L1 bands?
PTM · UniProt lists phosphoserine at 94, 98, 104, 153, 329, 343 and 747, and phosphotyrosine at 557. These are UniProt canonical-sequence coordinates; antibody or paper numbering may differ. Their presence does not demonstrate a visible mobility shift or explain the 68 kDa versus 86.4 kDa difference.
For a phospho-specific antibody, match its stated residue and numbering convention to the listed UniProt sites. Compare its signal with a total-AFAP1L1 blot and report the phospho signal relative to total signal. The supplied features identify sites but do not establish which are occupied in a given sample.
For a phospho-specific antibody, match its stated residue and numbering convention to the listed UniProt sites. Compare its signal with a total-AFAP1L1 blot and report the phospho signal relative to total signal. The supplied features identify sites but do not establish which are occupied in a given sample.
Does this guide establish induction of AFAP1L1?
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 AFAP1L1?
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 A11242 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 AFAP1L1 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.
Why might AFAP1L1 appear at 68 kDa instead of 86.4 kDa?
Interpretation · The reported apparent band is 68 kDa, while the predicted mass of the 768-residue canonical protein is 86.4 kDa. UniProt lists four isoforms, including ones with large sequence deletions, but these features alone do not identify the 68 kDa band. Check which isoforms the antibody can recognize before assigning it.
First compare each band with the predicted 86.4 kDa canonical protein and the sequence changes in isoforms 2–4. Then check whether the antibody epitope is retained in each candidate isoform. UniProt also lists phosphorylation, but neither that annotation nor isoform presence alone proves a band’s identity or mobility shift.
First compare each band with the predicted 86.4 kDa canonical protein and the sequence changes in isoforms 2–4. Then check whether the antibody epitope is retained in each candidate isoform. UniProt also lists phosphorylation, but neither that annotation nor isoform presence alone proves a band’s identity or mobility shift.
Boster reagents
AFAP1L1 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-AFAP1L AFAP1L1 Antibody
Cat # A11242
The catalog reports one anti-AFAP1L1 antibody, A11242, with reported Human and Mouse reactivity. Its Western blot image shows A549 cell lysate tested at 1 μg/mL; the supplied evidence does not show a Mouse sample.
Which to pick: A11242 is the only listed option and has a Western blot image using A549 cell lysate. For Mouse samples, reactivity is reported, but no Mouse Western blot example is supplied.
Source: BosterBio AFAP1L1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q8TED9.
- Human Protein Atlas. AFAP1L1 tissue expression.
- PMC3085893 — target-verified WB comparison
- PMC10515434 — target-verified WB comparison
- PMC9835296 — target-verified WB comparison