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- Table of Contents
CBX4 / E3 SUMO-protein ligase CBX4 · Western blot design guide
Design a Western Blot for CBX4
Real validated CBX4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CBX4 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
CBX4 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 | ~61.4 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Bone marrow (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 | Blocking-peptide control | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated CBX4 Western Blot Protocols
The A03388 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human brain tissue lysate (catalog A03388) |
| 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 CBX4 in human brain tissue lysate with CBX4 antibody at 1 μg/ml in (A) the absence and (B) the presence of blocking peptide (catalog A03388) |
| Primary antibody | A03388 · 1 μg/ml (catalog A03388) |
| 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 CBX4 Western Blot Band Size?
CBX4 is predicted at 61.4 kDa; isoforms and documented modifications could affect migration, but no empirical band size or distinct migration pattern is supplied.
| Band near 61.4 kDa | Consistent with the predicted CBX4 mass; identity requires controls |
| Additional band at another position | Could reflect isoform 1 or 2; their migration is not supplied |
| Close doublet | Could reflect CBX4 modification or isoforms; distinct bands are not established |
| Band offset from 61.4 kDa | CBX4 has documented acetylation and phosphorylation sites, but their migration effects are unknown |
💡Expected CBX4 appearanceCBX4 has a predicted mass of 61.4 kDa, but no empirical band size is supplied; verify a candidate band with blocking peptide and other band-identity controls.
| Predicted CBX4 mass | 61.4 kDa provides the sequence-based reference, not a validated band position |
| Splice isoforms 1 and 2 | May differ in size; their masses and resolution on a blot are not supplied |
| Lysine 149 acetylation | Documented modification; its effect on apparent size is not established |
| Serine 182 and 467 phosphorylation | Documented modifications; their effect on apparent size is not established |
| Threonine 497 phosphorylation | Documented modification; its effect on apparent size is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CBX4 is nuclear and may be poorly represented in the sampled fraction | Check a nuclear extract and confirm extraction quality |
| Band higher than expected | Band identity or migration effect is unverified | Compare with blocking-peptide competition and an independent CBX4 antibody |
| Band lower than expected | An alternative isoform or protein breakdown is possible, but neither band size is established | Check sample handling and confirm identity with an independent CBX4 antibody |
| Multiple bands | Isoforms 1 and 2 exist, but distinct migration is unproven | Use blocking-peptide competition to assess which bands are CBX4-dependent |
| Weak or no signal | Nuclear CBX4 may be underrepresented or poorly extracted | Check nuclear extraction and antibody performance with a positive-control lysate |
| Fragments below expected size | Sample proteolysis is possible; no CBX4 cleavage product is listed | Prepare fresh lysate with protease inhibitors and confirm fragments with an independent antibody |
Experiment evidence
Western blot images showing CBX4 band
A03388 · CBX4
M03388 · CBX4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CBX4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CBX4 in Western blot, you can use bone marrow lysate, which HPA scores as high expression.
Positive control: Bone marrow (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: CBX4 is nuclear, so nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for CBX4
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 |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | High | Protein (IHC) | HPA → |
| Duodenum | 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 → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Low | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced CBX4 Western Blot Tips
Deeper troubleshooting and optimisation questions for CBX4, answered from its protein features.
How should CBX4 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 CBX4 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. Isoform 2 lacks canonical residues 127–396, so a lower molecular weight band could be consistent with isoform 2. Confirm which isoform an antibody recognizes before assigning a band; these features do not establish either isoform’s observed migration.
Which CBX4 modification sites matter when interpreting bands?
PTM · In canonical UniProt numbering, Lys149 has an alternate N6-acetyllysine annotation; Ser182 and Ser467 are phosphorylated; Thr497 is phosphorylated by HIPK2. Isoform 2 lacks the region containing canonical Lys149 and Ser182. Check the numbering convention before comparing these sites with antibody or paper coordinates.
UniProt annotates phosphorylation of canonical CBX4 Thr497 by HIPK2. If testing HIPK2-related conditions, compare CBX4 signal across matched samples and verify Thr497 phosphorylation with a suitable site-specific assay. The annotation alone does not predict a detectable Western-blot shift.
UniProt annotates phosphorylation of canonical CBX4 Thr497 by HIPK2. If testing HIPK2-related conditions, compare CBX4 signal across matched samples and verify Thr497 phosphorylation with a suitable site-specific assay. The annotation alone does not predict a detectable Western-blot shift.
Does this guide establish induction of CBX4?
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 CBX4?
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 A03388 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 CBX4 bands be quantified across samples?
Quantitation · Use the same band assignment across samples and establish whether the antibody detects isoform 1, isoform 2, or both. Because CBX4 is annotated in the nucleus and nuclear speckles, compare like-for-like sample preparations. Report separate bands separately until their identities are established.
Should CBX4 migrate at its predicted 61.4 kDa?
Interpretation · Use 61.4 kDa as the predicted mass for canonical CBX4. The supplied features do not give an observed band position. Acetylation and phosphorylation are annotated, but their presence alone does not establish a visible shift or explain a difference from 61.4 kDa.
Check whether its size and antibody epitope are compatible with isoform 2, which lacks canonical residues 127–396. The supplied features do not identify an observed band or establish the identity of any lower band, so confirm the assignment experimentally.
CBX4 is annotated in the nucleus and nuclear speckles and as a component of a PRC1-like complex. Include a nuclear preparation when assessing detection, and keep preparation methods consistent when comparing samples. These annotations do not specify how much CBX4 a particular preparation will recover.
Check whether its size and antibody epitope are compatible with isoform 2, which lacks canonical residues 127–396. The supplied features do not identify an observed band or establish the identity of any lower band, so confirm the assignment experimentally.
CBX4 is annotated in the nucleus and nuclear speckles and as a component of a PRC1-like complex. Include a nuclear preparation when assessing detection, and keep preparation methods consistent when comparing samples. These annotations do not specify how much CBX4 a particular preparation will recover.
Boster reagents
CBX4 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-E3 SUMO-protein ligase CBX4 CBX4 Antibody
Cat # A03388
Real WB data
Anti-CBX4 Rabbit Monoclonal Antibody
Cat # M03388
The catalog reports two anti-CBX4 antibodies for WB, both with reported human, mouse, and rat reactivity. A03388 has a human brain lysate blot with and without blocking peptide. M03388 has blots from human cell lines, rat C6 cells, and mouse samples; its reported band is approximately 50 kDa versus 61 kDa expected.
Which to pick: Choose A03388 for the documented human brain lysate and blocking peptide comparison. Choose M03388 if its tested human cell lines, rat C6 cells, or mouse samples better match yours; account for the reported 50 kDa band.
Source: BosterBio CBX4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O00257.
- Human Protein Atlas. CBX4 tissue expression.
- PMC10568837 — target-verified WB comparison
- PMC6933416 — target-verified WB comparison
- PMC9828005 — target-verified WB comparison