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- Table of Contents
FZD4 / Frizzled-4 · Western blot design guide
Design a Western Blot for FZD4
Real validated FZD4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-FZD4 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
FZD4 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 | ~59.9 kDa | |
| Observed band | ~60 kDa | |
| Gel | 10% (catalog A02191-1) | |
| Positive control | Caudate (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 | Glycosylated + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated FZD4 Western Blot Protocols
The A02191-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HepG2, human PC-3, human K562 (catalog A02191-1) |
| Gel % | 10% (catalog A02191-1) |
| Load | 30 ug; reducing conditions (catalog A02191-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02191-1) |
| Membrane | nitrocellulose membrane (catalog A02191-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02191-1) |
| Primary antibody | A02191-1 · 0.5 μg/mL (catalog A02191-1) |
| Primary incubation | overnight at 4°C (catalog A02191-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02191-1) |
| Secondary incubation | 1.5 hour at RT (catalog A02191-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02191-1) |
| Detection | ECL (catalog A02191-1) |
Section 2
What Is the Expected FZD4 Western Blot Band Size?
FZD4 predicts 59.9 kDa and shows an empirical ~60 kDa band; the cause of any apparent difference is not established.
| Band near ~60 kDa | Matches the empirical FZD4 band; confirm identity with controls |
| Band below ~60 kDa | Could reflect cleavage of the 1–36 signal peptide; identity requires confirmation |
| Band above ~60 kDa | Could reflect N-linked glycosylation at Asn59 or Asn144; a visible shift is unproven |
| Weak or absent band in a soluble fraction | Membrane-localized FZD4 may be poorly recovered |
💡Expected FZD4 appearanceFZD4 has a predicted full-length mass of 59.9 kDa and an empirical band near 60 kDa; confirm band identity with a positive control and FZD4 depletion.
| Predicted full-length mass | 59.9 kDa, close to the empirical ~60 kDa band |
| N-linked glycosylation at Asn59 | May affect apparent mass; no visible shift is established |
| N-linked glycosylation at Asn144 | May affect apparent mass; no visible shift is established |
| Signal peptide at residues 1–36 | Cleavage makes the mature protein smaller than the full-length precursor; its migration is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-localized FZD4 may be poorly recovered | Check membrane extraction and run a positive-control lysate |
| Band higher than expected | N-linked glycosylation may affect migration | Compare with a deglycosylated sample and verify FZD4 identity |
| Band lower than expected | Signal-peptide cleavage is possible | Check antibody epitope and verify the band with FZD4 depletion |
| Broad smear instead of sharp band | Heterogeneous glycosylation is possible but unproven | Compare deglycosylated and untreated samples |
| Multiple bands | Processing or glycosylation may contribute; distinct forms are unproven | Compare reducing samples and use FZD4 depletion to identify specific bands |
| Weak or no signal | Incomplete recovery of membrane-localized FZD4 is possible | Check membrane enrichment and include a positive control |
Experiment evidence
Western blot images showing FZD4 band
A02191-1 · FZD4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for FZD4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for FZD4 in Western blot, you can use caudate tissue lysate, which HPA rates Medium for FZD4.
Positive control: Caudate (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside.
⚠️Feasibility: As a multi-pass membrane protein, FZD4 may require effective membrane-protein extraction for a detectable signal.
HPA tissue expression evidence for FZD4
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 |
|---|---|---|---|---|
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
| Cervix | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Medium | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Gallbladder | 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 → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Epididymis | glandular cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FZD4 Western Blot Tips
Deeper troubleshooting and optimisation questions for FZD4, answered from its protein features.
How should FZD4 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.
Could annotated isoforms explain multiple FZD4 bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. Do not label additional bands as annotated FZD4 isoforms on this evidence alone; assess their identity experimentally.
Which glycosylation sites matter when interpreting FZD4 bands?
PTM · UniProt lists N-linked glycosylation at Asn59 and Asn144, using full-length UniProt sequence numbering. If comparing treated and untreated samples, keep those sites in mind, but do not assign a band difference to glycosylation without experimental evidence.
Does this guide establish induction of FZD4?
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 FZD4 Western blot?
Transfer · FZD4 is a 537-residue, multi-pass cell-membrane protein. Check transfer efficiency for the ~60 kDa region when optimizing your membrane-protein workflow. The supplied features do not specify a transfer method or settings.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02191-1 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 can FZD4 membrane localization affect quantitation?
Quantitation · FZD4 is annotated at the cell membrane as a multi-pass protein. Keep membrane extraction and sample preparation consistent across samples, and quantify bands within the assay's linear range. Differences in recovered membrane protein can otherwise complicate comparisons.
How should the ~60 kDa band compare with predicted FZD4 mass?
Interpretation · The observed ~60 kDa band is close to the predicted 59.9 kDa. FZD4 has a signal peptide at UniProt residues 1–36 and two N-linked glycosylation sites, but these features alone do not establish a visible shift or account for a precise mass difference.
Consider band identity before assigning a cause. FZD4 has two listed N-linked sites, eight disulfide bonds, and one annotated isoform, but those features do not identify an unexpected band. Compare appropriate controls and sample preparation conditions.
UniProt lists eight disulfide bonds. Record whether samples were reduced and prepared consistently when comparing band patterns. The annotation alone does not predict a particular band position or prove that a band difference reflects disulfide bonding.
Consider band identity before assigning a cause. FZD4 has two listed N-linked sites, eight disulfide bonds, and one annotated isoform, but those features do not identify an unexpected band. Compare appropriate controls and sample preparation conditions.
UniProt lists eight disulfide bonds. Record whether samples were reduced and prepared consistently when comparing band patterns. The annotation alone does not predict a particular band position or prove that a band difference reflects disulfide bonding.
Boster reagents
FZD4 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-Frizzled 4/FZD4 Antibody Picoband®
Cat # A02191-1
The catalog reports A02191-1, an anti-FZD4 antibody with reported human, mouse, and rat reactivity. Its WB image shows an approximately 60 kDa band in human cell, rat lung, and mouse kidney lysates under the stated conditions. No orthogonal validation is supplied.
Which to pick: A02191-1 is the only listed option. Its WB image includes human HepG2, PC-3, K562, and RT4 cells, rat lung, and mouse kidney; use those tested samples and the reported conditions to judge its fit for your experiment.
Source: BosterBio FZD4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9ULV1.
- Human Protein Atlas. FZD4 tissue expression.
- PMC11413851 — target-verified WB comparison
- PMC8434704 — target-verified WB comparison
- PMC12865002 — target-verified WB comparison