WNT5B / Protein Wnt-5b · Western blot design guide

Design a Western Blot for WNT5B

Real validated WNT5B Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-WNT5B 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

Western blot protocol sheet for WNT5B: expected band ~40.3 kDa, hero antibody A05836, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **WNT5B** Western blot protocol sheet — expected band ~40.3 kDa, antibody A05836, controls and PMC citations. Open the full WNT5B WB guide →

Navigate to: Protocols Band Controls Antibodies

WNT5B 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	~40.3 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	Blocking peptide control
	Gene-set association	MSigDB Hallmark membership
	Isoform	1 isoform(s)

Section 1

Real Curated WNT5B Western Blot Protocols

The A05836 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.

Recommended Western blot protocol parameters
Sample / lysate	Jurkat cell lysate (catalog A05836)
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	Western blot analysis of WNT5 in Jurkat cell lysate with WNT5 antibody at 1 μg/ml in (A) the absence and (B) the presence of blocking peptide (catalog A05836)
Primary antibody	A05836 · 1 μg/ml (catalog A05836)
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 WNT5B Western Blot Band Size?

WNT5B has a predicted precursor mass of 40.3 kDa; signal-peptide cleavage and N-linked glycosylation may affect migration, but no empirical band or visible effect is established.

What am I looking at on my blot?
Band near 40.3 kDa	consistent with the predicted precursor mass; confirm WNT5B identity
Band below 40.3 kDa	could reflect removal of the 1–17 signal peptide; migration is unverified
Band above 40.3 kDa	could reflect N-linked glycosylation at Asn93, Asn99, Asn291, or Asn305; a visible shift is unverified
Little or no band in whole-cell lysate	consistent with secretion into the extracellular space or matrix

💡Expected WNT5B appearanceWNT5B has a predicted precursor mass of 40.3 kDa, but no empirical band size is supplied; signal-peptide cleavage and N-linked glycosylation could affect migration, so confirm band identity with appropriate controls.

How each factor affects band size
Predicted precursor mass	40.3 kDa is the sequence-based reference, not a measured band
Signal peptide at residues 1–17	cleavage produces a smaller mature chain, with no measured migration supplied
N-linked glycosylation at Asn93	could increase apparent mass if occupied; no shift is established
N-linked glycosylation at Asn99	could increase apparent mass if occupied; no shift is established
N-linked glycosylation at Asn291	could increase apparent mass if occupied; no shift is established
N-linked glycosylation at Asn305	could increase apparent mass if occupied; no shift is established

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	WNT5B is secreted into extracellular space and matrix	check conditioned medium or extracellular matrix fractions alongside lysate
Band higher than expected	N-linked glycosylation could alter migration	compare untreated and deglycosylated samples and confirm identity with WNT5B knockdown
Band lower than expected	the 1–17 signal peptide is cleaved	compare precursor and mature forms where available and confirm identity with WNT5B knockdown
Broad smear instead of sharp band	heterogeneous occupancy of N-linked glycosylation sites is possible	compare untreated and deglycosylated samples
Multiple bands	precursor, mature, or differently glycosylated forms are possible	compare fractions and deglycosylated samples, then confirm bands with WNT5B knockdown

Source: UniProt Q9H1J7 Human Protein Atlas product datasheet (A05836)

Experiment evidence

Western blot images showing WNT5B band

A05836 · WNT5B

M05836 · WNT5B

A05836-2 · WNT5B

← swipe / scroll for more images →

Real WB data behind the band-size expectations in this section.

Sample controls for WNT5B Western blot

🧪For positive controls for WNT5B in Western blot, you can use no HPA-supported tissue or cell sample because none is 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: WNT5B is secreted, so conditioned medium may be more informative than whole-cell lysate, but the supplied HPA data cannot establish tissue controls.

HPA tissue expression evidence for WNT5B

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 WNT5B Western Blot Tips

Deeper troubleshooting and optimisation questions for WNT5B, answered from its protein features.

How should WNT5B 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.

Are multiple WNT5B bands explained by listed isoforms?

Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple bands. Assess the bands alongside WNT5B's listed glycosylation sites, signal peptide, and disulfide bonds without assigning identities from those features alone.

Which WNT5B glycosylation sites matter when assessing a band?

PTM · UniProt lists N-linked glycosylation at Asn93, Asn99, Asn291, and Asn305. These are coordinates in the supplied 359-residue UniProt sequence. Glycosylation may affect migration, but the listed sites do not establish that a visible shift will occur.

Does this guide establish induction of WNT5B?

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 WNT5B?

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 A05836 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.

Which sample fraction matters for WNT5B quantitation?

Quantitation · WNT5B is listed as secreted and located in extracellular space and extracellular matrix. Specify whether quantitation uses cell lysate, extracellular material, or both, and compare like fractions across samples. A lysate band alone may not represent total WNT5B in the system.

Why might WNT5B migrate differently from its predicted 40.3 kDa?

Interpretation · The 40.3 kDa prediction is for the full-length protein. UniProt lists a signal peptide at residues 1–17 and four N-linked glycosylation sites, but these features alone cannot establish an apparent band size or explain a measured difference. No observed band size was supplied.

WNT5B has a signal peptide at residues 1–17 in the supplied UniProt sequence. Account for that region when comparing a full-length mass prediction with a processed protein. The features do not give an observed band size.

UniProt lists 11 disulfide bonds. Keep reducing conditions consistent when comparing lanes, since disulfide-dependent structure can affect migration. The feature count does not predict a specific band pattern or shift.

Check them against the supplied features: a 1–17 signal peptide, N-linked sites at Asn93, Asn99, Asn291, and Asn305, and 11 disulfide bonds. Coordinates refer to the supplied UniProt sequence; antibody or paper numbering may differ. These features suggest possibilities but cannot identify an unexpected band on their own.

Boster reagents

WNT5B 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 Western blot analysis of WNT5 in Jurkat cell lysate with WNT5 antibody at 1 μg/ml in (A) the absence and (B) the presence of blocking peptide.

Western blot analysis of WNT5 in Jurkat cell lysate with WNT5 antibody at 1 μg/ml in (A) the absence and (B) the presence of blocking peptide.

Anti-WNT5 WNT5B Antibody

Cat # A05836

View product →

Real WB data

Western blot analysis of WNT5B using anti-WNT5B antibody (M05836). Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human 293T whole cell lysates, Lane 2: human SW620 whole cell lysates. 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-WNT5B antigen affinity purified monoclonal antibody (M05836) at 1:500 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 ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for WNT5B at approximately 45 kDa. The expected band size for WNT5B is at 40 kDa.

Anti-Wnt5b Rabbit Monoclonal Antibody

Cat # M05836

View product →

Real WB data

Western blot analysis of Wnt5b using anti-Wnt5b antibody (A05836-2). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions. Lane 1: human HEK293 whole cell lysates, Lane 2: human SW620 whole cell lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-Wnt5b antigen affinity purified polyclonal antibody (Catalog # A05836-2) 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 1:10000 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 Wnt5b at approximately 45KD. The expected band size for Wnt5b is at 40KD.

Anti-Wnt5b Antibody Picoband®

Cat # A05836-2

View product →

Three the supplier antibodies have WB images: A05836 in Jurkat lysate with a blocking peptide, M05836 in 293T and SW620 lysates, and A05836-2 in HEK293 and SW620 lysates. The latter two report bands near 45 kDa versus 40 kDa expected; evidence is limited to these examples.

Which to pick: For human WB, choose M05836 or A05836-2 based on the closest reported lysate and protocol. A05836 lists Human, Mouse, and Rat reactivity, but its WB image uses Jurkat lysate; the supplied evidence does not show mouse or rat WB samples.

Source: BosterBio WNT5B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry Q9H1J7.
Human Protein Atlas. WNT5B tissue expression.
PMC13233589 — target-verified WB comparison
PMC12223781 — target-verified WB comparison
PMC2930277 — target-verified WB comparison