This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
INCENP / Inner centromere protein · Western blot design guide
Design a Western Blot for INCENP
Real validated INCENP Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-INCENP 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
INCENP 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 | ~105.4 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Appendix (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 | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated INCENP Western Blot Protocols
The A02466 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HEK293T, sp2/0, PC12 (catalog A02466) |
| 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 | A02466; 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 INCENP Western Blot Band Size?
INCENP is predicted at 105.4 kDa; isoforms and phosphorylation could affect migration, but no empirical band or visible shift is established.
| Band near 105.4 kDa | Consistent with the predicted INCENP size; confirm identity with controls |
| Single band away from 105.4 kDa | An alternative isoform is possible, but its migration is unknown |
| Two bands at different positions | Isoforms 1 and 2 are possible contributors; distinct migration is unverified |
| Closely spaced bands | Different phosphorylation states are possible; a visible shift is unverified |
💡Expected INCENP appearanceUniProt predicts INCENP at 105.4 kDa, but no empirical band size is supplied; confirm a candidate band with INCENP knockdown or another band-identity control.
| UniProt predicted mass | Places the reference size at 105.4 kDa |
| Isoform 1 | Has no supplied isoform-specific mass or migration |
| Isoform 2 | Has no supplied isoform-specific mass or migration |
| Alternative splicing of isoforms 1 and 2 | Could change band size, but distinct bands are not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | INCENP localizes to the nucleus, centromere, spindle and midbody | Check extraction and compare relevant cell fractions |
| Band higher than expected | Phosphorylation may affect mobility, but a shift is unverified | Compare phosphatase-treated material and confirm band identity |
| Band lower than expected | Isoform migration is unknown | Check isoform expression and confirm band identity by INCENP knockdown |
| Multiple bands | Isoforms or phosphorylation states are possible contributors | Use INCENP knockdown and compare phosphatase-treated material |
| Weak or no signal | Recovery may vary with INCENP's subcellular distribution | Check extraction and enrich the relevant fraction |
Experiment evidence
Western blot images showing INCENP band
A02466 · INCENP
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for INCENP Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for INCENP in Western blot, you can use appendix tissue, which HPA scores High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: INCENP is intracellular, and HPA identifies adipose tissue as a not-detected negative comparator.
HPA tissue expression evidence for INCENP
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 | endocrine cells | High | Protein (IHC) | HPA → |
| Colon | endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | endocrine cells | High | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | High | Protein (IHC) | HPA → |
| Rectum | endocrine 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 → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Not detected | Protein (IHC) | HPA → |
| Epididymis | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced INCENP Western Blot Tips
Deeper troubleshooting and optimisation questions for INCENP, answered from its protein features.
Where should the INCENP band appear relative to its predicted mass?
Band shift · The canonical sequence has a predicted mass of 105.4 kDa. No observed band position is supplied, so use 105.4 kDa as a reference rather than an expected apparent mass. The listed features alone cannot establish a visible shift or explain a difference from the prediction.
Could INCENP isoforms produce separate bands?
Isoforms · UniProt lists isoforms 1 and 2. Isoform 2 lacks residues 532–535 of the canonical sequence. Check which isoform an antibody can recognize, but do not assign separate bands from this four-residue difference without supporting evidence.
Which phosphorylation sites matter when choosing a phospho-specific antibody?
PTM · UniProt lists 37 modified residues, including Thr892, Ser893 and Ser894 phosphorylated by AURKB and AURKC. Match the antibody’s target site to the listed UniProt coordinate; paper or antibody numbering may use a different convention. A site annotation does not establish how strongly a band will appear.
INCENP has numerous listed phosphoserine and phosphothreonine sites, including Thr892, Ser893 and Ser894. These features support considering phosphorylation when examining band patterns, but they do not demonstrate a visible shift. Compare matched samples and confirm any proposed phosphorylation-dependent pattern experimentally.
INCENP has numerous listed phosphoserine and phosphothreonine sites, including Thr892, Ser893 and Ser894. These features support considering phosphorylation when examining band patterns, but they do not demonstrate a visible shift. Compare matched samples and confirm any proposed phosphorylation-dependent pattern experimentally.
Does this guide establish induction of INCENP?
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 INCENP?
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 A02466 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 can INCENP bands be quantified across samples?
Quantitation · Define the band being measured consistently across samples, and document whether the antibody detects total INCENP or a listed phosphorylation site. Because two isoforms and many phosphorylation sites are annotated, a phospho-specific signal should not automatically be treated as total INCENP abundance.
How should cell-cycle context affect INCENP comparisons?
Interpretation · INCENP is annotated for the cell cycle and mitosis and localizes to centromeres, the spindle and the midbody. Record and match the cell-cycle context when comparing samples. The supplied features do not specify an induction condition or predict a change in total INCENP abundance.
Check the band against the 105.4 kDa canonical prediction, the four-residue deletion in isoform 2 and the listed phosphorylation sites. None of these features alone identifies an unexpected band or proves a mass shift. Verify band identity before labeling additional bands as an isoform or modified INCENP.
Check the band against the 105.4 kDa canonical prediction, the four-residue deletion in isoform 2 and the listed phosphorylation sites. None of these features alone identifies an unexpected band or proves a mass shift. Verify band identity before labeling additional bands as an isoform or modified INCENP.
Boster reagents
INCENP 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-Inner centromere protein INCENP Antibody
Cat # A02466
A02466 is an anti-INCENP antibody listed for human, mouse, and rat. Its Western blot image uses HEK293T, sp2/0, and PC12 cell lysates at 1:500 dilution. No publication evidence is supplied.
Which to pick: A02466 is the only listed option. It has a Western blot image with three named cell lysates; use the reported samples and 1:500 dilution as the practical starting point.
Source: BosterBio INCENP gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9NQS7.
- Human Protein Atlas. INCENP tissue expression.
- PMC1783653 — target-verified WB comparison
- PMC4742097 — target-verified WB comparison