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- Table of Contents
WNT3A / Protein Wnt-3a · Western blot design guide
Design a Western Blot for WNT3A
Real validated WNT3A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-WNT3A 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
WNT3A 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 | ~39.4 kDa | |
| Observed band | 39 kDa | |
| Gel | 12–15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Glycosylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated WNT3A Western Blot Protocols
The PA2120 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Target-positive lysate and matched negative control (standard starting point) |
| 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 | PA2120; 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 WNT3A Western Blot Band Size?
WNT3A has a 39.4 kDa predicted precursor mass and a reported 39 kDa band; the cause of their small difference is not established.
| Band near 39 kDa | Empirical WNT3A band, close to the 39.4 kDa predicted precursor mass; confirm identity with antibody controls |
| Smaller band | Could reflect cleavage of the residues 1–18 signal peptide; its migration is not supplied |
| Higher band or broad smear | Could reflect variable N-linked glycosylation at Asn87 and Asn298; a visible effect is not established |
| Little or no band in whole-cell lysate | WNT3A is secreted into the extracellular space and matrix |
| Several bands | Isoforms 1 and 2 could differ in size, but distinct bands are not established |
💡Expected WNT3A appearanceThe predicted precursor mass is 39.4 kDa, while an antibody QC blot reports a 39 kDa band in whole-cell lysate; confirm band identity with appropriate antibody controls.
| Predicted precursor mass | 39.4 kDa; the reported empirical band is 39 kDa |
| N-linked glycosylation at Asn87 | Could increase apparent mass or broaden migration if occupied; no visible effect is established |
| N-linked glycosylation at Asn298 | Could increase apparent mass or broaden migration if occupied; no visible effect is established |
| Signal peptide at residues 1–18 | Cleavage could make mature WNT3A smaller than the precursor; its band size is not supplied |
| Splice isoforms 1 and 2 | Could differ in size; their masses and migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | WNT3A is secreted and may be scarce in whole-cell lysate | Check conditioned medium or an extracellular fraction alongside a positive control |
| Band higher than expected | N-linked glycosylation could affect migration, but the cause is unconfirmed | Compare with the 39 kDa reference and verify antibody specificity |
| Band lower than expected | Signal-peptide cleavage could reduce mass, but the cause is unconfirmed | Check epitope coverage and compare with a verified WNT3A control |
| Broad smear instead of sharp band | Variable occupancy of the two N-linked sites is possible but unproven | Compare samples with and without N-glycan removal and verify band identity |
| Multiple bands | Isoforms 1 and 2 could contribute, but distinct migration is unproven | Use isoform-aware controls or an independent antibody to identify the bands |
| Weak or no signal | Secreted WNT3A may be depleted from the sampled lysate | Check conditioned medium and include a verified positive control |
Experiment evidence
Western blot images showing WNT3A band
PA2120 · WNT3A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for WNT3A Western blot
🧪For positive controls for WNT3A in Western blot, you can use no HPA-supported positive tissue or cell sample because none is provided.
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 such as Ponceau alongside the samples.
⚠️Feasibility: WNT3A is secreted, so conditioned medium may give a stronger signal than whole-cell lysate; HPA supplies no tissue controls.
HPA tissue expression evidence for WNT3A
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 WNT3A Western Blot Tips
Deeper troubleshooting and optimisation questions for WNT3A, answered from its protein features.
How should WNT3A 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.
How might WNT3A isoforms affect the blot?
Isoforms · Two isoforms are listed. In isoform 2, the canonical lysine at UniProt position 352 is replaced by KNPGSRAGNSAHQPPHPQPPVRFHPPLRRAGKVP. Check whether the antibody recognizes the altered C-terminal sequence before interpreting different bands as isoforms.
Could WNT3A glycosylation affect band interpretation?
PTM · UniProt lists N-linked glycosylation at positions 87 and 298. When comparing bands, account for these sites, but do not assign a particular band to a glycosylated form from migration alone. Positions use UniProt sequence numbering.
Does this guide establish induction of WNT3A?
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 WNT3A?
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 PA2120 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 secreted WNT3A be quantified across samples?
Quantitation · Because WNT3A is secreted and associated with extracellular matrix, compare equivalent sample fractions and collection conditions across samples. Quantitation from a single fraction reflects WNT3A detected there, not necessarily the total produced.
Why is the observed WNT3A band near its predicted mass?
Interpretation · The reported band is 39 kDa, close to the 39.4 kDa predicted mass. WNT3A has a signal peptide at residues 1–18 and N-linked glycosylation sites at UniProt positions 87 and 298. These features alone do not establish a visible shift or explain the close match.
WNT3A is listed as secreted and present in extracellular space and extracellular matrix. Consider both conditioned medium and matrix-associated material when selecting samples; a cell lysate alone may not represent all WNT3A in the system.
UniProt lists 11 disulfide bonds. Record whether samples were run under reducing or nonreducing conditions, and compare conditions when investigating unexpected bands. The feature count alone does not predict a specific migration pattern.
WNT3A forms a soluble 1:1 complex with AFM that prevents oligomerization. If a band is unexpected, consider whether the sample contains AFM and how it was prepared. The complex annotation does not establish that a particular complex band will survive Western blot preparation.
WNT3A is listed as secreted and present in extracellular space and extracellular matrix. Consider both conditioned medium and matrix-associated material when selecting samples; a cell lysate alone may not represent all WNT3A in the system.
UniProt lists 11 disulfide bonds. Record whether samples were run under reducing or nonreducing conditions, and compare conditions when investigating unexpected bands. The feature count alone does not predict a specific migration pattern.
WNT3A forms a soluble 1:1 complex with AFM that prevents oligomerization. If a band is unexpected, consider whether the sample contains AFM and how it was prepared. The complex annotation does not establish that a particular complex band will survive Western blot preparation.
Boster reagents
WNT3A 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-Protein Wnt-3a Wnt3a Antibody Picoband®
Cat # PA2120
PA2120 is listed for human and mouse WNT3A. Its Western blot image shows SMMC whole-cell lysate (40 µg) tested at 0.5 µg/mL, with an observed 39 kDa band matching the predicted size. This documents one sample and condition.
Which to pick: PA2120 is the only listed antibody. Choose it if its reported SMMC lysate Western blot condition fits your experiment; the single image does not establish performance across other samples.
Source: BosterBio WNT3A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P56704.
- Human Protein Atlas. WNT3A tissue expression.
- PMC12626562 — target-verified WB comparison
- PMC8403793 — target-verified WB comparison
- PMC5349896 — target-verified WB comparison