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TOP2B / DNA topoisomerase 2-beta · Western blot design guide
Design a Western Blot for TOP2B
Real validated TOP2B Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TOP2B 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
TOP2B 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 | ~183.3 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
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 | 2 isoform(s) |
Section 1
Real Curated TOP2B Western Blot Protocols
The A02750-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | A549 (20ug), HEK293T (20ug), AML-12 (20ug) (catalog A02750-1) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A02750-1; 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 TOP2B Western Blot Band Size?
TOP2B has a predicted monomer mass of 183.3 kDa; isoforms and homodimer formation could affect band patterns, but their migration is unestablished.
| Band near 183.3 kDa | Consistent with the predicted TOP2B monomer; confirm identity with appropriate controls |
| Band near twice the monomer size | Could reflect a TOP2B homodimer that remains associated during analysis |
| Multiple bands near the expected region | Beta-1 and Beta-2 are possible contributors if they migrate differently; their separation is unknown |
| Band enriched in a nuclear fraction | Consistent with TOP2B localization in the nucleus |
💡Expected TOP2B appearanceUniProt predicts a 183.3 kDa TOP2B monomer, but no empirical band size is supplied; confirm any band with appropriate identity controls because isoform migration and dimer persistence are unestablished.
| UniProt predicted monomer mass | Sets the full-length reference at 183.3 kDa |
| TOP2B homodimer | Could appear near twice the monomer size if it remains associated during analysis |
| Beta-1 isoform | May differ in size from Beta-2, but its mass and migration are unspecified |
| Beta-2 isoform | May differ in size from Beta-1, but its mass and migration are unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear TOP2B may be poorly recovered | Check nuclear extraction and include a nuclear fraction |
| Band higher than expected | A TOP2B homodimer may remain associated | Compare thoroughly denatured samples and verify band identity |
| Band lower than expected | The band may be a fragment or a different protein | Verify identity by TOP2B depletion and compare antibody epitopes |
| Multiple bands | Beta-1 and Beta-2 may contribute, though distinct migration is unproven | Compare isoform expression and verify bands by TOP2B depletion |
| Weak or no signal | Nuclear TOP2B may be underrepresented in the sample | Check sample loading and enrich the nuclear fraction |
| Fragments below expected size | TOP2B may have degraded during sample preparation | Prepare fresh lysate with protease inhibitors and verify fragment identity |
Experiment evidence
Western blot images showing TOP2B band
A02750-1 · TOP2B
A02750 · TOP2B
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TOP2B Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TOP2B in Western blot, you can use appendix tissue lysate, which has high HPA expression.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: No tissue is listed as not detected, so use TOP2B knockdown or a KO line for a clear negative control.
HPA tissue expression evidence for TOP2B
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 | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Caudate | glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Colon | glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced TOP2B Western Blot Tips
Deeper troubleshooting and optimisation questions for TOP2B, answered from its protein features.
What band size should I expect for TOP2B?
Band shift · The supplied sequence predicts 183.3 kDa. No empirical apparent band size is provided, so use 183.3 kDa as a reference, not a confirmed migration position. The annotated modifications alone do not explain any difference between calculated and observed mass.
Could TOP2B isoforms produce nearby bands?
Isoforms · UniProt lists Beta-1 and Beta-2. Beta-1 lacks residues 24–28 relative to the supplied sequence. That five-residue difference could affect apparent size, but the features do not establish whether a Western blot can resolve the isoforms. Check whether the antibody recognizes the deleted region before assigning bands.
Which phosphorylation sites matter when interpreting TOP2B bands?
PTM · UniProt annotates many sites, including phosphoserine 1236, phosphothreonine 1292, phosphotyrosine 1370, 1421 and 1609, and further sites through serine 1613. These are UniProt coordinates; antibody or paper numbering may differ. Matched phosphatase-treated and untreated samples can test a suspected phosphorylation-related mobility change, but site annotation alone does not establish a visible shift.
UniProt lists N-acetylalanine at position 2 and N6-acetyllysine at position 3, using its sequence coordinates. These annotations establish modification sites, not a detectable Western-blot shift. Do not assign an unexpected band to acetylation without supporting experimental evidence.
The supplied UniProt features list no glycosylation sites, signal peptide or propeptide. They therefore provide no basis for assigning a higher band to glycosylation or precursor processing. Investigate the band experimentally rather than attributing its position to those mechanisms.
UniProt lists N-acetylalanine at position 2 and N6-acetyllysine at position 3, using its sequence coordinates. These annotations establish modification sites, not a detectable Western-blot shift. Do not assign an unexpected band to acetylation without supporting experimental evidence.
The supplied UniProt features list no glycosylation sites, signal peptide or propeptide. They therefore provide no basis for assigning a higher band to glycosylation or precursor processing. Investigate the band experimentally rather than attributing its position to those mechanisms.
Does this guide establish induction of TOP2B?
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 TOP2B Western blot?
Transfer · TOP2B is predicted to be 183.3 kDa. Use a transfer setup validated for proteins around that size, and check transfer with a high-mass marker or membrane stain before interpreting a weak signal. The supplied features do not identify a uniquely optimal transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02750-1 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 should TOP2B 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.
How should I compare TOP2B abundance across samples?
Interpretation · TOP2B is annotated in the nucleus, nucleolus and nucleoplasm. Keep the sample fraction consistent across comparisons and check that nuclear protein recovery is comparable. If measuring total TOP2B, confirm that the antibody detects both annotated isoforms; Beta-1 lacks residues 24–28.
First compare it with the 183.3 kDa predicted mass and the expected five-residue difference in Beta-1. TOP2B is annotated as a homodimer and has numerous modifications, but neither annotation identifies an unexpected band on its own. Test the assignment with an antibody against another region or a TOP2B depletion control.
First compare it with the 183.3 kDa predicted mass and the expected five-residue difference in Beta-1. TOP2B is annotated as a homodimer and has numerous modifications, but neither annotation identifies an unexpected band on its own. Test the assignment with an antibody against another region or a TOP2B depletion control.
Boster reagents
TOP2B 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-Topo II Beta (A32) TOP2B Antibody
Cat # A02750-1
Real WB data
Anti-TOP2B/Topoisomerase Ii Beta Antibody
Cat # A02750
Two the supplier anti-TOP2B antibodies have WB images: A02750-1 shows A549, HEK293T, AML-12, and PC12 lysates at 1:500; A02750 shows Jurkat lysate with a peptide-blocked lane. These images document the stated samples, not broad validation across species.
Which to pick: For human, mouse, or rat samples, consider A02750-1, whose WB image includes four named cell lysates. A02750 lists human and mouse reactivity and has a Jurkat WB image with peptide blocking. Match your sample species and desired evidence to the product.
Source: BosterBio TOP2B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q02880.
- Human Protein Atlas. TOP2B tissue expression.
- PMC13213696 — target-verified WB comparison
- PMC13312502 — target-verified WB comparison
- PMC9712534 — target-verified WB comparison