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- Table of Contents
CLIC4 / Chloride intracellular channel protein 4 · Western blot design guide
Design a Western Blot for CLIC4
Source-linked CLIC4 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CLIC4 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
CLIC4 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~28.8 kDa | |
| Observed band | ~29 kDa | |
| Gel | 5–20% (catalog M03210) | |
| Positive control | Colon (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 + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked CLIC4 Western Blot Protocol Options
The M03210 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human U251, human A431, human U20S, rat kidney, rat brain, mouse kidney, mouse brain (catalog M03210) |
| Gel % | 5–20% (catalog M03210) |
| Load | 30 ug; reducing conditions (catalog M03210) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M03210) |
| Membrane | nitrocellulose membrane (catalog M03210) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M03210) |
| Primary antibody | M03210 · 1:500 (catalog M03210) |
| Primary incubation | overnight at 4°C (catalog M03210) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:500 (catalog M03210) |
| Secondary incubation | 1.5 hour at RT (catalog M03210) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M03210) |
| Detection | ECL (catalog M03210) |
Section 2
What Is the Expected CLIC4 Western Blot Band Size?
CLIC4 is predicted at 28.8 kDa and observed at ~29 kDa; the supplied evidence does not establish a cause for the small difference.
| Band at ~29 kDa in whole-cell lysate | CLIC4 band consistent with the empirical observation and 28.8 kDa predicted mass |
| Band at ~29 kDa in a cytoskeletal fraction | consistent with CLIC4 cytoskeletal localization |
| Band at ~29 kDa in a membrane fraction | consistent with CLIC4 membrane localization |
| Band at ~29 kDa in a nuclear fraction | consistent with CLIC4 nuclear localization |
💡Expected CLIC4 appearanceCLIC4 has a predicted mass of 28.8 kDa and an observed band at ~29 kDa; confirm band identity with an appropriate positive control and CLIC4 depletion.
| UniProt predicted mass | 28.8 kDa predicted; the observed band is ~29 kDa |
| Ala2 N-acetylation | recorded modification with no demonstrated migration shift |
| Lys24 and Lys130 acetylation | recorded modifications with no demonstrated migration shift |
| Ser4, Ser132, Ser167, Ser236 and Tyr244 phosphorylation | recorded sites with no demonstrated migration shift |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CLIC4 may be poorly recovered from its cytoskeletal or membrane-associated pools | check fraction recovery and include a positive-control lysate |
| Band higher than expected | the supplied features do not establish a higher-migrating CLIC4 form | compare with the ~29 kDa control band and test antibody specificity by CLIC4 depletion |
| Band lower than expected | the supplied features do not establish a cleaved CLIC4 form | check sample integrity and test whether the band decreases after CLIC4 depletion |
| Broad smear instead of sharp band | no supplied feature establishes a CLIC4 smear | check sample quality and compare with a positive-control lysate |
| Multiple bands | additional bands are not explained by documented CLIC4 isoforms | identify the ~29 kDa band with a positive control and CLIC4 depletion |
| Weak or no signal | CLIC4 recovery or abundance in the sampled fraction may be low | check loading and compare whole-cell lysate with relevant enriched fractions |
Experiment evidence
Western blot images showing CLIC4 band
M03210 · CLIC4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CLIC4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CLIC4 in Western blot, you can use colon tissue lysate, which HPA scores High.
Positive control: Colon (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: HPA lists adipose tissue as not detected, making a tissue negative feasible, though its absence of signal should be verified.
HPA tissue expression evidence for CLIC4
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 |
|---|---|---|---|---|
| Colon | endothelial cells | High | Protein (IHC) | HPA → |
| Kidney | proximal tubules (microvilli) | High | Protein (IHC) | HPA → |
| Rectum | endothelial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Medium | Protein (IHC) | HPA → |
| Epididymis | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | endocrine cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CLIC4 Western Blot Tips
Deeper troubleshooting and optimisation questions for CLIC4, answered from its protein features.
How should CLIC4 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 another CLIC4 isoform explain a second band?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. Do not assign a second band to a CLIC4 isoform on this evidence alone.
Which CLIC4 modifications matter when interpreting bands?
PTM · UniProt lists phosphoserine at positions 4, 132, 167 and 236; phosphotyrosine at 244; N6-acetyllysine at 24 and 130; and N-acetylalanine at 2. These are UniProt sequence coordinates, which may differ from paper or antibody numbering. Their presence does not establish a visible band shift.
Does this guide establish induction of CLIC4?
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 CLIC4 Western blot?
Transfer · CLIC4 is a 253-residue protein predicted at 28.8 kDa. Choose a transfer setup suited to a roughly 29 kDa target, and check both membrane signal and residual protein in the gel when optimizing transfer.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M03210 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 CLIC4 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Should CLIC4 migrate above its predicted mass?
Interpretation · CLIC4 is predicted at 28.8 kDa, and the supplied observed band is about 29 kDa. Its listed modifications do not, by themselves, establish a visible shift or explain a mass difference.
CLIC4 is listed in cytoplasmic, membrane-associated and nuclear locations. Compare like fractions across samples and normalize within each fraction; a change in one fraction alone need not represent a change in total CLIC4.
Yes. UniProt lists CLIC4 at the cytoskeleton, cytoplasmic vesicle membrane, cell membrane and nucleus. A lysate or fraction that samples these compartments differently can change the detected signal. Record the fraction or extraction method when comparing lanes.
The supplied record lists no signal peptide, propeptide, glycosylation sites or alternative isoform. It also places CLIC4 in a multimeric cytoskeletal complex. These features alone do not identify an unexpected band; confirm its identity before assigning it to processing, modification or a complex.
CLIC4 is listed in cytoplasmic, membrane-associated and nuclear locations. Compare like fractions across samples and normalize within each fraction; a change in one fraction alone need not represent a change in total CLIC4.
Yes. UniProt lists CLIC4 at the cytoskeleton, cytoplasmic vesicle membrane, cell membrane and nucleus. A lysate or fraction that samples these compartments differently can change the detected signal. Record the fraction or extraction method when comparing lanes.
The supplied record lists no signal peptide, propeptide, glycosylation sites or alternative isoform. It also places CLIC4 in a multimeric cytoskeletal complex. These features alone do not identify an unexpected band; confirm its identity before assigning it to processing, modification or a complex.
Boster reagents
CLIC4 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-CLIC4 Rabbit Monoclonal Antibody
Cat # M03210
the supplier M03210 is a rabbit monoclonal anti-CLIC4 antibody listed for human, mouse, and rat. Its WB image shows an approximately 29 kDa band in the specified human cell and rodent tissue lysates. No publication evidence is supplied.
Which to pick: M03210 is the only listed option and has a WB image. Its caption reports testing in human 293T, U251, A431, and U20S cells and rat and mouse kidney and brain tissue, using 30 µg lysate and a 1:500 primary dilution.
Source: BosterBio CLIC4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9Y696.
- Human Protein Atlas. CLIC4 tissue expression.
- PMC7487930 — target-verified WB comparison
- PMC4354924 — target-verified WB comparison
- PMC3099427 — target-verified WB comparison