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- Table of Contents
TOPBP1 / DNA topoisomerase 2-binding protein 1 · Western blot design guide
Design a Western Blot for TOPBP1
Real validated TOPBP1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TOPBP1 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
TOPBP1 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 | ~170.7 kDa | |
| Observed band | ~170 kDa | |
| Gel | 8% (catalog A01867-1) | |
| Positive control | Adipose tissue (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 + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated TOPBP1 Western Blot Protocols
The A01867-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human HT1080, human HEL (catalog A01867-1) |
| Gel % | 8% (catalog A01867-1) |
| Load | 30 ug; reducing conditions (catalog A01867-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01867-1) |
| Membrane | nitrocellulose membrane (catalog A01867-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01867-1) |
| Primary antibody | A01867-1 · 0.5 μg/mL (catalog A01867-1) |
| Primary incubation | overnight at 4°C (catalog A01867-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01867-1) |
| Secondary incubation | 1.5 hour at RT (catalog A01867-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01867-1) |
| Detection | ECL (catalog A01867-1) |
Section 2
What Is the Expected TOPBP1 Western Blot Band Size?
TOPBP1 is predicted at 170.7 kDa and observed at ~170 kDa; the supplied features do not establish a cause for the small difference.
| Single band at ~170 kDa | matches the empirical TOPBP1 band and its 170.7 kDa predicted mass |
| ~170 kDa band in a nuclear fraction | fits TOPBP1 nuclear localization |
| Weak ~170 kDa band in a cytoplasmic fraction | may reflect its predominantly nuclear localization |
| Additional band near ~170 kDa | could reflect a modified form, but the listed phosphorylation sites do not establish a visible shift |
💡Expected TOPBP1 appearanceTOPBP1 has a predicted mass of 170.7 kDa and an empirical band at ~170 kDa in whole-cell lysate; verify band identity with antibody and sample controls.
| Predicted TOPBP1 mass | places the full-length protein near 170.7 kDa, consistent with the observed ~170 kDa band |
| Phosphothreonine at residue 298 | may affect mobility, but a visible shift is unproven |
| Phosphoserine at residue 301 | may affect mobility, but a visible shift is unproven |
| Phosphothreonine at residue 779 | may affect mobility, but a visible shift is unproven |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | nuclear TOPBP1 may be poorly recovered | check nuclear extraction and a positive whole-cell lysate |
| Band higher than expected | a modified form is possible, but its identity is unestablished | compare with the ~170 kDa band and confirm identity using an independent antibody |
| Band lower than expected | the supplied features do not establish a smaller mature form | check sample integrity and confirm identity with an independent antibody |
| Multiple bands | modified forms are possible, but distinct migration is unproven | compare band patterns with a positive control and an independent antibody |
| Weak or no signal | limited recovery of nuclear TOPBP1 is possible | check extraction and include a positive whole-cell lysate |
Experiment evidence
Western blot images showing TOPBP1 band
A01867-1 · TOPBP1
A01867-1 · TOPBP1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TOPBP1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TOPBP1 in Western blot, you can use adipose tissue.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: No tissue is reported as not detected, so use siRNA knockdown or a KO line as the negative control.
HPA tissue expression evidence for TOPBP1
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 |
|---|---|---|---|---|
| Adipose tissue | adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | adipocytes | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Liver | cholangiocytes | Medium | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced TOPBP1 Western Blot Tips
Deeper troubleshooting and optimisation questions for TOPBP1, answered from its protein features.
How should TOPBP1 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 a second band represent a listed TOPBP1 isoform?
Isoforms · Only one isoform is supplied, with no alternative sequence. These features do not support assigning a second band to a specific TOPBP1 isoform.
Which TOPBP1 phosphorylation sites matter when interpreting bands?
PTM · UniProt lists phosphothreonine at 298, 779, 848, 861, 1062, and 1064, and phosphoserine at 301, 860, 864, 886, 888, 1002, and 1504. These are UniProt coordinates; antibody or paper numbering may differ. Site annotation alone does not establish a visible band shift.
Does this guide establish induction of TOPBP1?
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 TOPBP1 Western blot?
Transfer · TOPBP1 is predicted at 170.7 kDa. Use transfer conditions suitable for a protein of that size, and check that protein near ~170 kDa reached the membrane. The supplied features do not specify a transfer method or settings.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A01867-1 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.
What should be considered when quantifying TOPBP1?
Quantitation · TOPBP1 is annotated in the nucleus and on chromosomes, with recruitment to DNA damage sites during mitosis. Compare like fractions across samples and normalize within the fraction measured; a change in one fraction need not represent a change in total protein.
How should the ~170 kDa band compare with predicted TOPBP1 mass?
Interpretation · The observed band is ~170 kDa, close to the predicted 170.7 kDa. The listed phosphorylation sites do not, by themselves, establish a visible shift or explain any small difference in apparent mass.
TOPBP1 is linked to DNA damage and repair, and its interaction with phosphorylated MDC1 promotes recruitment to DNA damage sites during mitosis. Recruitment does not establish an increase in total TOPBP1 abundance. Interpret a signal change in light of the fraction measured and its normalization.
Compare them with the ~170 kDa observed band and 170.7 kDa predicted mass. The supplied record lists one isoform, 13 phosphorylation sites, and no glycosylation sites. Those annotations alone cannot identify an unexpected band or show that phosphorylation caused a shift.
TOPBP1 is linked to DNA damage and repair, and its interaction with phosphorylated MDC1 promotes recruitment to DNA damage sites during mitosis. Recruitment does not establish an increase in total TOPBP1 abundance. Interpret a signal change in light of the fraction measured and its normalization.
Compare them with the ~170 kDa observed band and 170.7 kDa predicted mass. The supplied record lists one isoform, 13 phosphorylation sites, and no glycosylation sites. Those annotations alone cannot identify an unexpected band or show that phosphorylation caused a shift.
Boster reagents
TOPBP1 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-TOPBP1 Antibody Picoband®
Cat # A01867-1
The catalog reports A01867-1 as an anti-TOPBP1 antibody with reported human, mouse, and rat reactivity. Its Western blot image shows an approximately 170 kDa band in human HeLa, HT1080, and HEL whole cell lysates. The supplied image does not show mouse or rat samples.
Which to pick: A01867-1 is the only listed option. Choose it if its reported reactivity fits your sample; the supplied Western blot image documents testing in three human cell lysates at 0.5 μg/mL.
Source: BosterBio TOPBP1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q92547.
- Human Protein Atlas. TOPBP1 tissue expression.
- PMC3358587 — target-verified WB comparison
- PMC4701836 — target-verified WB comparison
- PMC6486426 — target-verified WB comparison