NUMA1 Antibody, Biotin conjugated
Description
Molecular Composition and Design
The NUMA1 Antibody, Biotin conjugated comprises:
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Target Epitope: Recognizes human NUMA1 (UniProt ID: Q14980), a 238 kDa nuclear matrix protein involved in mitotic spindle organization and asymmetric cell division .
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Conjugation: Covalently linked to biotin via lysine or cysteine residues, enabling binding to streptavidin-enzyme complexes (e.g., HRP) .
2.1. Immunoassays
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ELISA: Used in sandwich ELISA kits (e.g., Human NUMA1 ELISA Kit) with:
| Parameter | Value | Source |
|---|---|---|
| Assay Type | Sandwich ELISA | |
| Sample Types | Serum, plasma, tissue lysates | |
| Incubation Time | 3.5 hours |
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Western Blot: Detects NUMA1 at 238 kDa in lysates (e.g., HeLa cells) , with recommended dilutions of 1:500–1:5,000 .
2.2. Functional Studies
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Cancer Research: NUMA1 deletion reduces breast cancer stem cell (BCSC) viability and metastasis in triple-negative breast cancer (TNBC) models .
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Mitotic Regulation: NUMA1-KO cells exhibit resistance to BMI1 inhibition, implicating NUMA1 in spindle pole integrity and cell cycle progression .
3.1. In Vitro Studies
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Biotinylation Efficiency: Co-expression of bacterial biotin ligase (BirA) enhances biotinylation of NUMA1-BAP fusions by >10×, improving detection sensitivity .
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Specificity: No cross-reactivity with NUMA1 homologs or unrelated proteins confirmed via epitope mapping .
3.2. Preclinical Models
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TNBC Therapy: NUMA1 knockdown in BF3M mouse mammary tumor cells reduces ALDH+ and CD29hiCD61+ BCSCs, suppressing tumorigenesis and lung metastasis .
4.2. Recommended Dilutions
| Application | Dilution Range | Source |
|---|---|---|
| ELISA | 1:500–1:2,000 | |
| Western Blot | 1:500–1:5,000 | |
| Immunofluorescence | 1:50–1:200 |
Comparative Analysis of Biotin-Conjugated vs. Other Formats
| Feature | Biotin Conjugate | HRP Conjugate | FITC Conjugate |
|---|---|---|---|
| Detection Method | Streptavidin-enzyme | Direct chromogenic | Fluorescence |
| Signal Amplification | High (biotin-streptavidin) | Moderate | Low |
| Multiplexing | Compatible | Limited | Yes |
| Cost | $166–$458 | $166–$458 | $166–$458 |
Source: Product data from Cloud-Clone and Assay Genie .
Future Directions
Product Specs
Target Background
Nuclear mitotic apparatus protein 1 (NuMA1) is a microtubule (MT)-binding protein essential for spindle pole formation, maintenance, chromosome alignment, and segregation during mitosis. It anchors the minus ends of MTs at the spindle poles, crucial for spindle pole establishment and stability. NuMA1 plays a vital role in mitotic spindle orientation during metaphase and elongation during anaphase, processes dependent on dynein-dynactin. Specifically, during metaphase, NuMA1 is part of a ternary complex (GPSM2 and G(i) alpha proteins) regulating dynein-dynactin recruitment and anchoring to the mitotic cell cortex above the spindle poles, ensuring accurate spindle orientation. In anaphase, NuMA1, through direct interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), recruits and accumulates the dynein-dynactin complex at the polar cortical cell membrane, regulating spindle elongation and chromosome segregation. In vitro studies demonstrate NuMA1 binding to other polyanionic phosphoinositides, including phosphatidylinositol 3-phosphate (PIP), lysophosphatidic acid (LPA), and phosphatidylinositol triphosphate (PIP3). NuMA1 is also critical for proper mitotic spindle orientation in asymmetric cell divisions, mitotic MT aster assembly, anastral spindle assembly, and positively regulates TNKS protein localization to spindle poles during mitosis. A highly abundant component of the nuclear matrix, NuMA1 may have a non-mitotic structural role, occupying a significant portion of the nuclear volume. It is also required for epidermal differentiation and hair follicle morphogenesis.
NuMA1's function is extensively documented in the literature. Key findings include:
- Galectin-3/NuMA interaction is functionally significant for spindle pole organization; glycosylation-mediated NuMA localization is required for spindle pole cohesion (PMID: 28469279).
- p37 negatively regulates PP1's function on NuMA, resulting in reduced cortical NuMA levels and proper spindle orientation (PMID: 29222185).
- E-cadherin regulates the assembly of the LGN/NuMA complex at cell-cell contacts, linking cell division orientation to intercellular adhesion (PMID: 28045117).
- NuMA targets dynactin to spindle microtubule minus-ends, localizing dynein activity (PMID: 29185983).
- Quantitative UBC analysis in urine samples identified a cutoff (≥6.7 ng/ml) for detecting bladder cancer (PMID: 28824318).
- A short NuMA isoform may function as a tumor suppressor (PMID: 28748856).
- NuMA plays a central role in cellular homeostasis through its involvement in rDNA transcription and p53-independent nucleolar stress response (PMID: 28981686).
- Importin-alpha/-beta regulates NuMA function in higher-order microtubule structure assembly, including the mitotic spindle (PMID: 28939615).
- Low NuMA post-translational modifications are associated with neoplasms (PMID: 28209915).
- Seven NuMA isoforms, generated by alternative splicing, are categorized into long, middle, and short groups; lower short NuMA expression is observed in cancer cells (PMID: 25451259).
- Fusion of KSHV LANA with NuMA enhances ori-P-containing plasmid replication (PMID: 27829174).
- The NuMA-Astrin interaction is crucial for accurate cell division (PMID: 27462074).
- Aurora-A regulates NuMA's dynamic exchange between cytoplasmic and spindle pole pools, phosphorylating NuMA's C-terminus and influencing its dynamic behavior and spindle orientation functions (PMID: 26832443).
- APC2 negatively regulates LGN localization at the cell cortex by competing with NuMA for binding (PMID: 26766442).
- Risk factors for false-positive urinary NMP22 results in bladder cancer detection (PMID: 24976592).
- At low-grade disease, NMP22 is more sensitive than urine cytology for detecting recurrent bladder urothelial carcinoma (PMID: 25488052).
- NuMA interacts with phosphoinositides, linking the mitotic spindle to the plasma membrane; correct anaphase NuMA localization is mediated by direct membrane phospholipid binding (PMID: 24996901).
- pRB regulates NuMA's mitotic function and spindle organization (PMID: 24350565).
- MAP velocity across microtubules affects frictional forces, with NuMA exhibiting lower friction when moving toward minus ends (PMID: 24725408).
- Mitosis-dependent SUMO-1 modification of NuMA contributes to mitotic spindle pole formation and maintenance (PMID: 24309115).
- NuMA ectopic expression manipulates p53 and p21 transcriptional expression during interphase (PMID: 23828576).
- BRAP2 ectopic expression inhibits nuclear localization of HMG20A and NuMA1, preventing nuclear envelope SYNE2 accumulation (PMID: 23707952).
- Hepatocyte Par1b and the LGN-NuMA complex define lumen position in epithelial cell division (PMID: 24165937).
- CDK1-mediated NuMA phosphorylation couples mitotic progression with cortical dynein function (PMID: 23921553).
- Aurora-A phosphorylation of NuMA is important for cell survival (PMID: 23097092).
- Dynein and astral microtubules mediate Galphai/LGN/NuMA complex transport from the cell cortex to spindle poles (PMID: 23389635).
- NuMA (nuclear mitotic apparatus protein) plays a role in upper tract urothelial tumors (PMID: 21865670).
- NuMA recruits cyclin-dependent kinase 8, influencing p53-mediated p21 gene function (PMID: 23589328).
- Inscuteable (Insc) and NuMA are mutually exclusive LGN interactors (PMID: 22977735).
- NuMA and Eg5 regulate spindle assembly (PMID: 23368718).
- NuMA expression is upregulated in tumors, associating with disease stage in mucinous EOC subtypes, lymph node involvement, and patient age (PMID: 22719996).
- Low NUMA1 is associated with glioblastoma (PMID: 22619067).
- NuMA is essential for microtubule connection to meiosis I spindle poles and organized early spindle assembly (PMID: 22552228).
- Apoptotic rearrangement of interchromatin granule clusters involves nuclear matrix (NuMA) rearrangement and chromatin association, depending on phosphatases, caspases, and CAD (PMID: 22023725).
- Urinary NMP22 and CK18 levels are significantly higher in patients with bladder transitional cell carcinoma compared to non-transitional cell carcinoma (PMID: 19615282).
- Accurate NuMA distribution is important for human oocyte maturation, zygote, and embryo development; proper NuMA assembly is necessary for bipolar spindle organization and oocyte developmental competence (PMID: 21297155).
- NuMA is expressed in interphase nuclei of fibroblasts and oocytes (PMID: 21406448).
- NuMA-RARalpha copy number correlates with phenotype onset in mice (PMID: 21255834).
- NuMA contributes to interphase nuclear structural support by organizing chromatin (PMID: 20467816).
- Ric-8A and Gi alpha recruit LGN, NuMA, and dynein to the cell cortex to orient the mitotic spindle (PMID: 20479129).
- pADPr's dynamic cross-linking function at spindle poles, involving PARP-5a and NuMA, promotes two-pole assembly (PMID: 19759176).
- A NuMA C-terminal domain interacts with tubulin, inducing microtubule bundling, stabilization, and abnormal mitotic spindle formation (PMID: 11956313).
- NuMA cleavage differs in Jurkat T and HeLa cells, indicating varying caspase activation; NuMA's intranuclear distribution changes during apoptosis (PMID: 12508117).
- NuMA's role in myelodysplastic leukemia with promyelocytic features (PMID: 14737102).
- Proteins with NuMA C-terminus-like regions are found in diverse vertebrate species (PMID: 15388855).
- Multiple mechanisms regulate NUMA1 dynamics at spindle poles (PMID: 15561764).
- NuMA gene variations may increase breast cancer risk (PMID: 15684076).
- NuMA's diverse roles in vertebrate cells (review) (PMID: 16146802).
- NuMA influences mammary epithelial differentiation by affecting chromatin organization (PMID: 17108325).
- The Rae1-NuMA interaction is critical for normal mitotic spindle formation (PMID: 17172455).
Q&A
What experimental validations are critical when implementing biotin-conjugated NUMA1 antibodies in immunofluorescence (IF) assays?
Methodological Answer:
Validation requires three parallel approaches:
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Specificity Verification: Perform siRNA-mediated NUMA1 knockdown followed by IF staining to confirm signal reduction .
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Cross-Reactivity Testing: Use Western blotting with lysates from NUMA1-knockout cell lines (e.g., HAP1 NUMA1-KO clones) to verify absence of off-target binding .
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Titration Optimization: Conduct checkerboard assays comparing antibody dilutions (1:100–1:1000) against positive controls (e.g., mitotic HeLa cells) .
Key Data:
| Validation Step | Critical Parameter | Reference |
|---|---|---|
| Specificity | ≥70% signal reduction post-knockdown | |
| Cross-reactivity | No bands in NUMA1-KO lysates | |
| Optimal dilution | 1:500 for nuclear signal clarity |
How does NUMA1’s role in mitotic spindle regulation impact experimental design in cancer models?
Advanced Mechanistic Context:
NUMA1 tethers spindle microtubules to centrosomes, with knockout studies showing:
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Mitotic Arrest Duration: NUMA1-WT cells exhibit 131 min arrest vs. 92 min in NUMA1-KO under BMI1 inhibition .
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Cell Fate Outcomes: Prolonged arrest in WT correlates with CyclinB1 degradation and apoptosis .
Experimental Design Considerations:
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Use live-cell imaging with FUCCI reporters to track cell cycle transitions.
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Combine with dynein inhibitors (e.g., Ciliobrevin D) to dissect NUMA1-dynein interactions .
What methodological pitfalls affect quantification in NUMA1 ELISA using biotin-conjugated antibodies?
Technical Challenges & Solutions:
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Matrix Effects: Heparin plasma shows 82–88% recovery vs. 93–101% in serum .
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Mitigation: Pre-dilute samples in zero standard buffer (1:4).
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Hook Effect: Observed at >200 ng/mL NUMA1 concentrations .
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Resolution: Perform serial dilutions for samples with unknown concentrations.
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Protocol Optimization Table:
| Parameter | Requirement | Impact on CV% |
|---|---|---|
| Wash Steps | ≥5 cycles | Reduces CV from 12% → 6% |
| Incubation Temp | 37°C ± 0.5°C | Prevents edge effects (ΔOD450 < 0.1) |
| TMB Exposure | ≤15 min | Avoids saturation (linear range: 0.1–2.0 OD) |
How to resolve contradictions in NUMA1 localization data between IHC and IF assays?
Discrepancy Analysis Framework:
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Artifact Check: Compare paraffin-embedded (IHC) vs. frozen (IF) sections. Nuclear matrix dissolution in FFPE may obscure interphase signals .
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Antibody Epitope Mapping: The biotin conjugate in PACO54033 targets AA 627–870 , while A02018-1 binds N-terminal epitopes .
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Quantitative Correlation: Use spatial transcriptomics to align protein and mRNA distributions.
What advanced applications exist for biotinylated NUMA1 antibodies beyond basic detection?
Innovative Methodologies:
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Proximity Ligation Assays (PLA): Identify NUMA1 interaction partners (e.g., dynein/dynactin) with <40 nm resolution .
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Multiplexed CLEM: Correlate light-level NUMA1 distribution with EM-based spindle architecture.
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Single-Cell Western Blotting: Resolve NUMA1 isoforms in mitotic subpopulations.
Validation Requirement Table:
| Application | Controls Needed | Reference Standard |
|---|---|---|
| PLA | NUMA1-KO cells + IgG isotype | Dynein IC2 knockout lysates |
| CLEM | Gold-standard anti-α-tubulin | EM tomograms of metaphase spindles |
How does NUMA1 antibody performance vary across tumor subtypes in IHC?
Tissue-Specific Optimization:
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Nuclear vs. Cytoplasmic Staining:
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Quantitative Thresholds: ≥30% nuclear positivity defines NUMA1-high status in NSCLC .
What computational tools enhance analysis of NUMA1 antibody-derived datasets?
Advanced Workflows:
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Spindle Pole Tracking: Use FIJI’s TrackMate with TMRM-stained mitochondria as fiducials.
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Multiplex Data Integration: Align IF signals with RNA-seq clusters via CODEX pipelines.
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Machine Learning: Train U-Net models to classify mitotic defects (lagging chromosomes, multipolar spindles) from NUMA1 images.
How to troubleshoot low signal in flow cytometry with biotin-NUMA1 antibodies?
Stepwise Debugging Protocol:
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Permeabilization Check: Compare 0.1% Triton vs. saponin protocols (saponin preserves epitope accessibility ).
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Biotin Saturation Test: Pre-block with 50 μg/mL free biotin for 30 min.
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Compensation Controls: Use NUMA1-KO cells spiked with 5% WT for threshold setting.
Signal Recovery Table:
| Issue | Corrective Action | Expected ΔMFI |
|---|---|---|
| Over-fixation | Reduce PFA from 4% → 2% | +150% |
| Insufficient blocking | Add 10% goat serum + 1% BSA | +80% |
| Suboptimal conjugation | Switch to streptavidin-PE (vs. FITC) | +200% |
