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- Table of Contents
CKAP4 / Cytoskeleton-associated protein 4 · Western blot design guide
Design a Western Blot for CKAP4
Source-linked CKAP4 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CKAP4 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
CKAP4 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 | ~66 kDa | |
| Observed band | ~70 kDa | |
| Gel | 10% (catalog A02154-2) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Heart muscle (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 Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked CKAP4 Western Blot Protocol Options
The A02154-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A431, human Hela, human HepG2, human U251, rat PC-12, rat RH35, mouse NIH/3T3, mouse HEPA1-6 (catalog A02154-2) |
| Gel % | 10% (catalog A02154-2) |
| Load | 30 ug; reducing conditions (catalog A02154-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02154-2) |
| Membrane | nitrocellulose membrane (catalog A02154-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02154-2) |
| Primary antibody | A02154-2 · 1:10000 (catalog A02154-2) |
| Primary incubation | overnight at 4°C (catalog A02154-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02154-2) |
| Secondary incubation | 1.5 hour at RT (catalog A02154-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02154-2) |
| Detection | ECL (catalog A02154-2) |
Section 2
What Is the Expected CKAP4 Western Blot Band Size?
CKAP4 is predicted at 66 kDa and observed near 70 kDa in reducing whole-cell blots; the cause of the difference is not established.
| Band near 70 kDa in whole-cell lysate | Matches the empirical CKAP4 band; confirm identity with a CKAP4 control. |
| Band near 66 kDa | Near the predicted CKAP4 mass; confirm identity. |
| Weak band in a soluble fraction | CKAP4 may remain in the membrane fraction. |
| Band enriched in a membrane fraction | Consistent with CKAP4 membrane localization; confirm identity. |
💡Expected CKAP4 appearanceUniProt predicts 66 kDa, while antibody QC reports a CKAP4 band near 70 kDa in reducing whole-cell lysates; confirm band identity with CKAP4 controls.
| UniProt predicted mass | Sets the 66 kDa sequence-based reference; the empirical band is near 70 kDa. |
| Phosphoserines at residues 3, 17, and 19 | Annotated modifications; their effect on apparent band size is not established. |
| Phosphoserines at residues 232 and 312 | Annotated modifications; their effect on apparent band size is not established. |
| N6-acetyllysine at residue 21 | Annotated modification; its effect on apparent band size is not established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated CKAP4 may be poorly extracted. | Check lysis conditions and test a membrane-enriched fraction. |
| Band higher than expected | The reported band is near 70 kDa, above the 66 kDa prediction; the cause is unestablished. | Compare with the reported band and verify identity by CKAP4 depletion. |
| Band lower than expected | The lower band's identity or integrity is uncertain. | Check sample integrity and verify identity by CKAP4 depletion. |
| Multiple bands | Additional band identities are uncertain; only one isoform is listed. | Use CKAP4 depletion to identify CKAP4-dependent bands. |
| Weak or no signal | Membrane extraction or transfer may limit detection. | Check extraction, loading, and transfer controls. |
| Fragments below expected size | Sample degradation is possible; no fragment size is established. | Check sample handling and confirm fragment identity by CKAP4 depletion. |
Experiment evidence
Western blot images showing CKAP4 band
A02154-2 · CKAP4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CKAP4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CKAP4 in Western blot, you can use adrenal gland lysate, which shows high expression in HPA.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Heart muscle (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: High expression in adrenal gland and no detected expression in heart muscle make tissue controls feasible.
HPA tissue expression evidence for CKAP4
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | endocrine cells | High | Protein (IHC) | HPA → |
| Cervix | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Colon | endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | paneth cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Lung | alveolar cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced CKAP4 Western Blot Tips
Deeper troubleshooting and optimisation questions for CKAP4, answered from its protein features.
How should CKAP4 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 CKAP4 isoform explain an unexpected band?
Isoforms · The supplied record lists one isoform and no alternative sequence. Do not assign another band to a CKAP4 isoform on this evidence alone; check antibody specificity and whether the band tracks with CKAP4 enrichment or depletion.
Which CKAP4 modifications matter when interpreting Western blots?
PTM · UniProt lists phosphoserines at positions 3, 17, 19, 232 (by FAM20C), and 312, plus N6-acetyllysine at 21. These are UniProt coordinates; reconcile any antibody or paper numbering before comparing sites. Their presence does not establish a resolvable band shift.
Does this guide establish induction of CKAP4?
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 CKAP4 Western blot?
Transfer · Start with a transfer workflow suitable for a protein near 66–70 kDa, then check the membrane and post-transfer gel for recovery. CKAP4 is a single-pass membrane protein found at the endoplasmic reticulum and cell membrane, so verify that sample preparation extracts it effectively. The supplied features do not establish a preferred transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02154-2 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 CKAP4 bands be quantified across samples?
Quantitation · Use comparable sample preparations and normalize CKAP4 signal to an appropriate loading measure. CKAP4 is reported at membranes, the cytoskeleton, and the perinuclear region, so changes in a particular fraction need not represent changes in total CKAP4. Keep exposure within a measurable range.
Why does CKAP4 appear near 70 kDa instead of 66 kDa?
Interpretation · The supplied predicted mass is 66 kDa, while the observed band is about 70 kDa. Use the observed position to guide band identification. The listed features alone do not establish why CKAP4 migrates differently or demonstrate a visible modification-dependent shift.
Compare them with the observed ~70-kDa band and test whether they follow CKAP4 enrichment or depletion. The supplied record lists one isoform, no alternative sequence, and no glycosylation sites. Its phosphorylation and acetylation annotations alone cannot identify an additional band or prove a shift.
Compare them with the observed ~70-kDa band and test whether they follow CKAP4 enrichment or depletion. The supplied record lists one isoform, no alternative sequence, and no glycosylation sites. Its phosphorylation and acetylation annotations alone cannot identify an additional band or prove a shift.
Boster reagents
CKAP4 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-CKAP4 Antibody Picoband®
Cat # A02154-2
A02154-2 is a rabbit polyclonal anti-CKAP4 antibody with a WB image showing a band near 70 kDa (expected 66 kDa) in the named human, rat, and mouse cell lysates. Evidence supplied here is limited to this product image and caption.
Which to pick: A02154-2 is the only listed option. Its reported reactivity is human, mouse, and rat, and its WB image includes A431, HeLa, HepG2, U251, PC-12, RH35, NIH/3T3, and HEPA1-6 whole cell lysates under reducing conditions.
Source: BosterBio CKAP4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q07065.
- Human Protein Atlas. CKAP4 tissue expression.
- PMC3320026 — target-verified WB comparison
- PMC12663859 — target-verified WB comparison
- PMC9883288 — target-verified WB comparison