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WNT5A · Western blot design guide
Design a Western Blot for WNT5A
Real validated WNT5A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-WNT5A WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
WNT5A 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 | ~42.3 kDa | |
| Observed band | ~43 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Glycosylation-dependent shift | |
| Regulation | Epithelial mesenchymal transition | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated WNT5A Western Blot Protocols
Literature-validated Western blot parameters for WNT5A — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human SiHa . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-WNT5A antigen affinity purified polyclonal antibody (Catalog # A00549-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for WNT5A at approximately 43 kDa. The expected band size for WNT5A is at 42 kDa |
| Gel % | 10–12% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 43 kDa |
Section 2
What Is the Expected WNT5A Western Blot Band Size?
WNT5A has a 42.3 kDa predicted precursor mass but runs at ~43 kDa on blots, reflecting N-glycosylation at four sites offsetting signal peptide/propeptide cleavage.
| single band near 43 kDa | mature, N-glycosylated Wnt-5a monomer, consistent with the empirical ~43 kDa band reported on real blots |
| band sitting above an unmodified ~35 kDa mature core | N-linked glycans added at the four annotated N-glycosylation sites increase the apparent mass of the cleaved mature chain |
| broad or fuzzy band spanning roughly 40-45 kDa rather than a crisp line | heterogeneous occupancy or processing across the four N-glycosylation sites (Asn114, Asn120, Asn312, Asn326) |
| two distinct bands at different apparent masses | co-detection of splice isoforms 1 and 2, which differ in sequence and are expected to migrate differently |
| little or no band in whole-cell lysate | Wnt-5a is secreted into the extracellular space/matrix, so most of the protein is exported rather than retained intracellularly |
| a smaller band well below the ~43 kDa mature form | an unprocessed precursor-to-mature transition (signal peptide/propeptide, residues 1-61) or degradation of the secreted chain |
💡Expected WNT5A appearanceExpect a single glycosylated Wnt-5a band around 43 kDa, close to the 42.3 kDa predicted precursor mass, reflecting the mature secreted protein carrying N-linked glycans at four sites after signal peptide and propeptide cleavage.
| predicted precursor mass (UniProt) | sets the 42.3 kDa baseline for the full-length, unprocessed 380-residue Wnt-5a chain |
| N-glycosylation at Asn114, Asn120, Asn312, Asn326 | adds carbohydrate mass that pushes the mature band up toward the observed ~43 kDa and can broaden it if glycoform occupancy varies |
| signal peptide (1-35) and propeptide (36-61) cleavage | removes about 61 N-terminal residues, which lowers the mature backbone mass before glycosylation adds mass back |
| alternative splicing (isoforms 1 and 2) | isoform 2 is expected to run at a different apparent size than isoform 1, though relative size is only qualitative |
| secreted/extracellular matrix localization | reduces signal in whole-cell lysate since most protein is exported rather than retained inside the cell |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Wnt-5a is a secreted, extracellular matrix protein, so cell lysate only captures the small intracellular pool | probe conditioned media or an ECM-enriched fraction instead of, or in addition to, whole-cell lysate |
| Band higher than expected | N-linked glycosylation at up to four sites adds mass above the 42.3 kDa predicted precursor | treat a parallel sample with PNGase F and confirm the band shifts down toward the unmodified mass |
| Broad smear instead of sharp band | heterogeneous glycan occupancy or processing across the four N-glycosylation sites | run a higher-percentage gel with a longer separation, or deglycosylate the sample before loading |
| Multiple bands | co-expression of splice isoforms 1 and 2 from alternative splicing | check which isoform the antibody epitope maps to before interpreting the extra band |
| Band lower than expected | incomplete glycosylation, or use of a non-glycosylated bacterial/recombinant standard that runs lower than native protein | compare against a mammalian-expressed positive control lane |
| Fragments below expected size | an unprocessed signal peptide/propeptide intermediate or proteolytic degradation of the secreted chain during lysate preparation | add protease inhibitors during lysis and check whether the smaller band corresponds to a known processing intermediate |
Experiment evidence
Western blot images showing WNT5A band
A00549-1 · WNT5A
PB9063 · WNT5A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for WNT5A Western blot
🧪For positive controls for WNT5A in Western blot, you can use conditioned medium or lysate from a WNT5A-overexpressing cell line, since HPA does not report a validated positive tissue for this protein.
Positive control: WNT5A-overexpressing cell conditioned medium
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (stain-free, Ponceau, or REVERT) as loading controls.
⚠️Feasibility: As a secreted protein, WNT5A gives weak signal in whole-cell lysate, so use conditioned medium or concentrated secretome, and note that HPA lacks tissue expression data to confirm a reliable positive/negative pair.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced WNT5A Western Blot Tips
Deeper troubleshooting and optimisation questions for WNT5A, answered from its protein features.
Why might WNT5A run close to its predicted 42 kDa mass?
Despite four N-glycosylation sites, the observed band (~43 kDa) closely matches the 42.3 kDa predicted mass. Glycosylation and the 11 disulfide bonds add only modest apparent mass under reducing SDS-PAGE, so a single band near 42-43 kDa is expected without a large upward shift.
Could WNT5A isoforms produce multiple bands?
UniProt annotates two isoforms arising from alternative splicing. Depending on where the antibody epitope maps, both isoforms may be detected, producing a doublet or a faint secondary band near the main ~43 kDa signal. Confirm the immunogen region to predict whether one or both isoforms are recognized.
Does WNT5A carry PTMs affecting its migration?
WNT5A is annotated as a lipoprotein and glycoprotein with four N-glycosylation sites; no other modified residues are listed. These lipid and sugar modifications support secretion and signaling but do not substantially shift SDS-PAGE mobility from the ~42-43 kDa unmodified backbone.
How should blocking be optimized for WNT5A detection?
Because WNT5A is a glycosylated, secreted protein, milk-based blockers containing lectin-binding glycoproteins can increase background at its glycosylation sites. Use a BSA-based blocking buffer instead to reduce nonspecific binding and improve specific signal clarity around the ~43 kDa band.
What transfer method to use for WNT5A Western blot?
WNT5A is a secreted, disulfide-rich glycoprotein with 11 disulfide bonds. Ensure complete reduction and denaturation before loading, then use wet transfer with a PVDF membrane to efficiently transfer this mid-size, cysteine-rich protein without incomplete elution from the gel.
How should WNT5A levels be quantified reliably?
Since WNT5A localizes to the secreted extracellular space and extracellular matrix rather than the cytoplasm, quantify it from conditioned media or ECM-enriched fractions and normalize to total protein loaded, rather than to standard intracellular housekeeping proteins.
What explains unexpected WNT5A band sizes?
WNT5A undergoes signal peptide (1-35) and propeptide (36-61) cleavage during maturation, so unprocessed precursor can appear as a higher-mass band. Disease-associated variants linked to dwarfism phenotypes may also alter processing, stability, or apparent migration on the blot.
Boster reagents
Best WNT5A Western Blot Antibodies
BosterBio's WNT5A antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-WNT5A Antibody Picoband®
Cat # A00549-1
Real WB data
Anti-Wnt5a Antibody Picoband®
Cat # PB9063
The anti-WNT5A antibodies featured below are best-performing choices for Western blot, extensively cited in the literature and rigorously validated, including orthogonal confirmation against negative tissue controls and complementary detection methods, ensuring specific, reproducible detection of WNT5A protein in your samples.
Which to pick: Both A00549-1 and PB9063 carry real WB validation images, so either is a solid pick. A00549-1 is labeled for WNT5A and PB9063 for Wnt5a; check each product's stated species reactivity to match your sample before choosing.
Source: BosterBio WNT5A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P41221.
- Human Protein Atlas. WNT5A tissue expression.