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- Table of Contents
CLDN4 / Claudin-4 · Western blot design guide
Design a Western Blot for CLDN4
Real validated CLDN4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CLDN4 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
CLDN4 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 | ~22.1 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (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 | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated CLDN4 Western Blot Protocols
The M03683 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | MCF7 cell lysate (catalog M03683) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M03683; 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 CLDN4 Western Blot Band Size?
Claudin-4 is predicted at 22.1 kDa; its membrane location, phosphorylation, disulfide, and claudin associations have no demonstrated migration effect here.
| Band near 22.1 kDa | Consistent with the predicted Claudin-4 monomer; confirm identity with controls. |
| Faint or absent band in a soluble lysate fraction | Claudin-4 is a multi-pass membrane protein that may remain in the membrane fraction. |
| No distinct phosphorylation doublet | Tyr208 phosphorylation is annotated, but a resolvable mobility shift is not established. |
| Band above 22.1 kDa | Could reflect retained claudin associations; band identity requires confirmation. |
💡Expected CLDN4 appearanceThe predicted Claudin-4 mass is 22.1 kDa; no empirical band size is supplied, and bands near that size still require ordinary identity controls.
| Predicted monomer mass | Provides a 22.1 kDa reference, not a validated migration position. |
| Tyr208 phosphorylation by EPHA2 | May alter apparent migration, but no visible shift is established. |
| Intrachain disulfide between Cys54 and Cys64 | May affect conformation under non-reducing conditions; it does not establish a doubled mass. |
| Heteropolymeric strands with other claudins | May produce higher bands if associations persist during preparation; no fixed band size is established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | This multi-pass membrane protein may be poorly recovered in a soluble lysate. | Check the membrane fraction and use membrane-compatible extraction with a positive control. |
| Band higher than expected | Retained claudin associations or an unrelated band are possible. | Check denaturation conditions and confirm identity with a second antibody or CLDN4 depletion. |
| Band lower than expected | A fragment or unrelated band is possible; no cleavage product is annotated. | Use protease inhibitors and confirm identity with CLDN4 depletion. |
| Weak or no signal | Incomplete recovery of membrane-associated Claudin-4 may reduce signal. | Check membrane extraction, sample loading, and a positive control. |
| Fragments below expected size | Proteolysis during sample handling is possible, but fragment sizes are not supplied. | Prepare fresh lysate with protease inhibitors and confirm fragments with an independent epitope antibody. |
Experiment evidence
Western blot images showing CLDN4 band
M03683 · CLDN4
A03683 · CLDN4
A03683 · CLDN4
A03683 · CLDN4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CLDN4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CLDN4 in Western blot, you can use appendix tissue, which HPA reports as highly expressing CLDN4.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Because CLDN4 is a multi-pass membrane protein, membrane-enriched lysate may improve detection.
HPA tissue expression evidence for CLDN4
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 |
|---|---|---|---|---|
| Appendix | endocrine cells | High | Protein (IHC) | HPA → |
| Colon | endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | endocrine cells | High | Protein (IHC) | HPA → |
| Kidney | distal tubules | High | Protein (IHC) | HPA → |
| Pancreas | exocrine glandular cells | High | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CLDN4 Western Blot Tips
Deeper troubleshooting and optimisation questions for CLDN4, answered from its protein features.
How should CLDN4 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 CLDN4 isoforms explain multiple bands?
Isoforms · Only one isoform is listed, with no alternative sequence. The supplied features therefore do not support assigning additional bands to CLDN4 isoforms.
Where is CLDN4 phosphorylated, and could this shift its band?
PTM · UniProt lists phosphotyrosine at position 208, modified by EPHA2. That coordinate uses the supplied 209-residue UniProt sequence; antibody or paper numbering may differ. The modification is a reason to investigate a band difference, but its presence alone does not establish a visible shift.
Does this guide establish induction of CLDN4?
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 CLDN4 Western blot?
Transfer · CLDN4 is a 22.1 kDa multi-pass membrane protein. A 0.2 µm membrane is a practical starting point to retain this small protein; check transfer efficiency and adjust conditions if signal is weak.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M03683 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 CLDN4 bands be quantified?
Quantitation · CLDN4 localizes to the cell membrane and tight junctions. Keep membrane extraction and sample preparation consistent, normalize to a suitable loading measure, and quantify bands within the assay's linear signal range. Different recovery of membrane protein can otherwise affect comparisons.
Should CLDN4 migrate at exactly 22.1 kDa?
Interpretation · 22.1 kDa is the predicted mass; no observed band position is supplied. CLDN4 is a multi-pass membrane protein, so use the predicted value as a guide rather than treating a different apparent mass as proof of another protein. Its listed features alone do not establish a visible shift.
CLDN4 can form strands with other claudins and has one listed disulfide bond. These features make sample preparation and reducing conditions relevant checks, but they do not establish that a higher Western-blot band is an oligomer. Compare reducing conditions and verify band identity before assigning it.
No signal peptide or propeptide is listed for the 209-residue CLDN4 sequence. The supplied features do not support explaining a smaller band by cleavage of either segment; verify any smaller band independently.
CLDN4 can form strands with other claudins and has one listed disulfide bond. These features make sample preparation and reducing conditions relevant checks, but they do not establish that a higher Western-blot band is an oligomer. Compare reducing conditions and verify band identity before assigning it.
No signal peptide or propeptide is listed for the 209-residue CLDN4 sequence. The supplied features do not support explaining a smaller band by cleavage of either segment; verify any smaller band independently.
Boster reagents
CLDN4 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-Claudin 4 Monoclonal Antibody
Cat # M03683
Real WB data
Anti-CLAUDIN4 CLDN4 Antibody
Cat # A03683
Two the supplier anti-CLDN4 antibodies are listed for Western blotting, both with human reactivity and WB images. M03683 shows an MCF7 cell lysate blot; A03683 reports human cell line blot conditions. No publication evidence is supplied.
Which to pick: For MCF7 lysate, M03683 has the directly named sample example. A03683 provides reported loading and antibody incubation conditions for human cell lines. Both list human reactivity and a WB image; choose based on the sample and conditions closest to your experiment.
Source: BosterBio CLDN4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O14493.
- Human Protein Atlas. CLDN4 tissue expression.
- PMC10105442 — target-verified WB comparison
- PMC7664319 — target-verified WB comparison
- PMC12281411 — target-verified WB comparison