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NDC80 · Western blot design guide
Design a Western Blot for NDC80
Real validated NDC80 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NDC80 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
NDC80 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 | ~73.9 kDa | |
| Observed band | ~74 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Coiled-coil mobility shift | |
| Regulation | G2m checkpoint | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated NDC80 Western Blot Protocols
Literature-validated Western blot parameters for NDC80 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human K562 , Lane 2: rat spleen , Lane 3: mouse spleen . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-NDC80 antigen affinity purified polyclonal antibody (Catalog # A01731-2) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for NDC80 at approximately 74KD. The expected band size for NDC80 is at 74KD |
| Gel % | 10–12% |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 74 kDa |
Section 2
What Is the Expected NDC80 Western Blot Band Size?
NDC80 has a 73.9 kDa predicted mass and runs at ~74 kDa, matching almost exactly since it lacks glycosylation, cleavage, or disulfide dimerization that would shift its size.
| single sharp band at ~74 kDa | matches the predicted 73.9 kDa mass of NDC80, since it carries no glycosylation, cleavage, or dimerization to shift its size |
| faint upward smear or doublet near 74-80 kDa in mitotic samples | cell-cycle phosphorylation, such as Ser165 by NEK2, can transiently retard migration during mitosis |
| no extra bands at other molecular weights | only one NDC80 isoform is annotated, so additional splice-variant bands are not expected |
| weaker signal in cytoplasm-only fractions versus nuclear/chromatin extracts | NDC80 is concentrated in the nucleus and at kinetochores, not distributed generally through the cytoplasm |
| no mobility change between reducing and non-reducing SDS-PAGE | NDC80 has no disulfide bonds, so reducing agents do not alter its migration |
💡Expected NDC80 appearanceExpect a single sharp band at the empirically observed ~74 kDa, matching NDC80's predicted 73.9 kDa mass almost exactly, since it carries no glycosylation, cleavage, or disulfide-linked dimerization to shift its apparent size.
| Predicted mass (73.9 kDa) | sets the baseline migration position, consistent with the observed ~74 kDa band |
| Absence of N-/O-glycosylation | keeps the band sharp and close to predicted mass rather than smeared or shifted upward |
| Absence of disulfide bonds | prevents any disulfide-linked higher-molecular-weight band from forming under non-reducing conditions |
| Cell-cycle phosphorylation (e.g., Ser165 by NEK2) | can cause a slight upward mobility shift or faint doublet in mitotic cell lysates |
| Single annotated isoform | means no additional splice-variant bands at other molecular weights are expected |
| Heterotetrameric NDC80 complex assembly (with CDCA1, SPBC24, SPBC25) | partners may co-purify under native conditions, but under standard reducing SDS-PAGE NDC80 still resolves as its monomeric ~74 kDa band |
| Situation | Likely cause | Next action |
|---|---|---|
| Weak or no signal | NDC80 is concentrated at kinetochores mainly from late prophase to anaphase, so asynchronous cultures have few positive cells | use mitotically enriched or synchronized (e.g., nocodazole-arrested) lysates and increase total protein loaded |
| No band in lysate | cytoplasmic-only fractionation misses the nuclear and kinetochore-localized pool of NDC80 | prepare nuclear or chromatin-enriched extracts rather than whole cytoplasmic lysate |
| Multiple bands | cell-cycle-regulated phosphorylation, such as NEK2-mediated Ser165 phosphorylation, produces a shifted or doublet species in mitotic cells | compare mitotic-arrested versus interphase lysates and treat a parallel sample with phosphatase to collapse the doublet |
| Band higher than expected | hyperphosphorylation during mitosis retards migration above the ~74 kDa expected position | confirm with a phosphatase-treated control lysate and note the cell-cycle stage of the sample |
| Band lower than expected | proteolytic degradation of the coiled-coil NDC80 protein during lysis generates smaller fragments | add fresh protease inhibitors and keep lysates cold throughout preparation |
Experiment evidence
Western blot images showing NDC80 band
A01731-2 · NDC80
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NDC80 Western blot
🧪For positive controls for NDC80 in Western blot, you can use lysates from actively dividing/proliferating cells (e.g. HeLa), since NDC80 is a mitotic kinetochore protein expressed specifically from late prophase to anaphase.
Positive control: Proliferating cells (e.g. HeLa)
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH, β-actin, and a total-protein stain (e.g. stain-free, Ponceau S, or REVERT) alongside as loading controls.
⚠️Feasibility: HPA provides no tissue/cell expression data for NDC80, and since its kinetochore localization is restricted to mitotic cells, controls should use synchronized/proliferating cultures plus siRNA knockdown or a KO line rather than a tissue-based negative control.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced NDC80 Western Blot Tips
Deeper troubleshooting and optimisation questions for NDC80, answered from its protein features.
Why does the observed band match the predicted mass so closely?
NDC80 has no signal peptide, propeptide, glycosylation, or disulfide bonds, so its mature molecular weight closely tracks the 73.9 kDa sequence-based prediction. The ~74 kDa observed band confirms a full-length, unmodified-mass protein with no proteolytic processing or heavy PTM burden altering migration.
Does NDC80 have isoforms that could cause extra bands?
UniProt lists only one isoform for NDC80, so multiple bands from alternative splicing are not expected. A single ~74 kDa band should represent the full-length protein; additional bands more likely reflect degradation, non-specific binding, or co-migrating complex partners rather than isoform diversity.
Why might NDC80 signal vary between cell cycle stages?
NDC80 is annotated with Cell cycle, Cell division, and Mitosis keywords, and localizes to kinetochores only from late prophase to anaphase (PubMed:14699129). Detection is therefore cell-cycle-dependent; unsynchronized cultures may show weak or inconsistent signal, while synchronizing cells in mitosis increases detectable kinetochore-associated protein.
Could phosphorylation shift NDC80's apparent molecular weight?
NDC80 carries nine modified residues, consistent with its Phosphoprotein and Acetylation keywords. Mitotic phosphorylation of kinetochore proteins can cause slight upward mobility shifts or faint doublets on SDS-PAGE. Comparing mitotic-arrested versus interphase lysates helps confirm whether a doublet reflects phosphorylation rather than a nonspecific band.
What blocking approach suits NDC80 detection?
NDC80 has no glycosylation sites, so glycoprotein-specific blocking precautions are unnecessary. Standard 5% non-fat milk or BSA in TBST is sufficient; BSA is preferable if the same membrane will later be probed with phospho-specific antibodies against NDC80's modified residues.
What transfer method to use for NDC80 Western blot?
At ~74 kDa, NDC80 transfers efficiently with standard wet or semi-dry transfer using 20% methanol Tris-glycine buffer. Its coiled-coil structure does not require the extended transfer times needed for very high molecular weight or membrane proteins; a standard 60-90 minute wet transfer is typically sufficient.
How should NDC80 levels be normalized for quantitation?
Because NDC80 expression and kinetochore localization are cell-cycle-regulated, synchronize cell populations before comparing samples and avoid normalizing to a loading control that is itself cell-cycle-dependent. Use a stable housekeeping protein and report synchronization stage or mitotic index alongside band intensity for accurate comparisons.
What could explain higher molecular weight NDC80 bands?
NDC80 forms the NDC80 complex with CDCA1, SPBC24, and SPBC25 through a coiled-coil interface. Incomplete denaturation of this tightly associated complex can leave residual higher-order species if samples are under-boiled or run non-reducing. Thorough boiling in SDS/reducing buffer should resolve a clean ~74 kDa monomeric band.
Boster reagents
Best NDC80 Western Blot Antibodies
BosterBio's NDC80 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-HEC1/HEC/NDC80 Antibody Picoband®
Cat # A01731-2
Our recommended anti-NDC80 antibody is Boster's best-performing option for Western blot, extensively cited in the literature and thoroughly validated, with orthogonal cross-validation against negative tissue controls and complementary methods confirming specificity for reliable, reproducible detection.
Which to pick: Only one Boster NDC80 antibody is listed, A01731-2, which includes an actual Western blot validation image, making it the clear, ready-to-use choice for your NDC80 WB experiments without needing to compare alternatives.
Source: BosterBio NDC80 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O14777.
- Human Protein Atlas. NDC80 tissue expression.