SPN Monoclonal Antibody
Description
Target Antigen and Molecular Identity
The SPN mAb binds to the NuMA protein, a high-molecular-mass component critical for spindle pole organization during mitosis . Key characteristics include:
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Molecular Weight: ~240 kDa
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Localization: Relocates from the interphase nucleus to spindle poles during mitosis
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Function: Facilitates microtubule organization and chromosomal segregation .
NuMA’s role in maintaining spindle integrity makes it a pivotal target for studying mitotic defects.
Mechanism of Action
SPN mAb disrupts NuMA’s function by binding to its epitopes, leading to:
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Micronuclei Formation: Post-mitotic cells exhibit fragmented nuclei due to failed chromosomal segregation .
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Cytokinesis Defects: Impaired spindle organization results in incomplete cell division.
Experimental microinjection of SPN-3 (a specific SPN mAb) into PtK2 cells revealed stage-dependent effects:
| Injection Stage | Abnormal Cytokinesis (%) | Micronuclei Formation (%) |
|---|---|---|
| Prophase | 90% | 90% |
| Prometaphase | 78% | 78% |
| Metaphase | 77% | 77% |
| Anaphase | 16% | 16% |
Data sourced from SPN-3 microinjection experiments in PtK2 cells .
Mitotic Studies
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SPN mAb has been used to model mitotic errors, mimicking effects of microtubule-targeting agents like colcemid and taxol .
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In HeLa cells, SPN mAb injection induced spindle disorganization comparable to pharmacological disruption .
Functional Insights
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Early Mitotic Dependency: NuMA is essential during prophase to metaphase but becomes dispensable by anaphase .
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Therapeutic Parallels: Defects induced by SPN mAb mirror those seen in cancer therapies targeting microtubules .
Comparative Analysis with Other Antibodies
SPN mAb’s effects differ from antibodies targeting bacterial pathogens (e.g., anti-PhtD mAbs for Streptococcus pneumoniae) . While SPN mAb focuses on eukaryotic cell mechanisms, anti-PhtD mAbs neutralize bacterial virulence factors.
| Antibody Type | Target | Application | Mechanism |
|---|---|---|---|
| SPN mAb | NuMA protein | Mitotic research | Disrupts spindle organization |
| PhtD3 + 7 | S. pneumoniae | Bacterial coinfection therapy | Opsonizes pathogens |
Future Directions
Product Specs
The SPN monoclonal antibody is produced by immunizing mice with a synthesized peptide derived from the human CD43 protein. Following immunization, B cells are isolated from the mouse and fused with myeloma cells to generate hybridomas. Hybridomas producing the SPN antibody are selected and cultured in the mouse abdominal cavity. The SPN monoclonal antibody is then affinity-purified from mouse ascites using affinity chromatography with a specific immunogen. It is suitable for ELISA and immunohistochemistry (IHC) applications to detect the human SPN protein.
SPN (sialophorin), also known as CD43 or leukosialin, primarily functions as a negative regulator of cell adhesion. It inhibits leukocyte adhesion to the endothelium and extracellular matrix proteins by preventing the binding of other adhesion molecules. This action inhibits leukocyte recruitment to sites of inflammation. Beyond its role in cell adhesion, CD43 has also been implicated in regulating lymphocyte activation and differentiation.
Target Background
CD43, a predominant cell surface sialoprotein of leukocytes, plays a crucial role in regulating multiple T-cell functions, including activation, proliferation, differentiation, trafficking, and migration. It positively regulates T-cell trafficking to lymph nodes through its association with ERM proteins (EZR, RDX, and MSN). CD43 negatively regulates Th2 cell differentiation, promoting the differentiation of T cells toward a Th1 lineage commitment. It enhances the expression of interferon-gamma (IFN-gamma) by T cells during T-cell receptor (TCR) activation of naïve cells and induces IFN-gamma expression by CD4(+) T cells and, to a lesser extent, by CD8(+) T cells.
CD43 is involved in preparing T cells for cytokine sensing and differentiation into effector cells by inducing the expression of cytokine receptors IFNGR and IL4R, promoting IFNGR and IL4R signaling, and mediating the clustering of IFNGR with TCR. It acts as a major E-selectin ligand, responsible for Th17 cell rolling on activated vasculature and recruitment during inflammation. CD43 mediates Th17 cells, but not Th1 cells, adhesion to E-selectin. It functions as a T-cell counter-receptor for SIGLEC1, protecting cells from apoptotic signals and promoting cell survival.
- CD43 expression was observed in 95.7% of atypical chronic lymphocytic leukemia cases. PMID: 28713070
- Most non-hematopoietic neoplasms exhibit negative CD43 expression. PMID: 28807337
- This study identified a novel signaling pathway for CD43 through the regulation of alternative functions of pyruvate kinase isoform M2, promoting cell survival following activation. PMID: 27606486
- Forty percent of adenoid cystic carcinomas showed staining for CD43, while no cases of basal cell carcinoma were positive. PMID: 25551301
- CD43 expression is a novel adverse prognostic factor for patients with diffuse large B-cell lymphomas. PMID: 25682152
- CD43 polymorphisms have been linked to tuberculosis susceptibility. PMID: 25078322
- Host membrane proteins PSGL-1, CD43, and CD44 interact with assembling HIV-1 Gag through polybasic sequences present in the cytoplasmic tails of the membrane proteins and in Gag. PMID: 25320329
- When used as a vaccine in mice, the 2/165 phagotope elicited antibodies against the UN1/CD43 antigen, suggesting that the 2/165 phagotope mimics the UN1 antigen structure and could represent a novel immunogen for cancer immunotherapy. PMID: 24356816
- CD43 promotes cell transformation by preventing merlin-mediated contact inhibition of growth. PMID: 24260485
- CD43 is an adverse prognostic marker in DLBCL and is preferentially expressed in the non-GCB subgroup. PMID: 23617469
- CD43 regulates the threshold for T cell activation by targeting Cbl functions. PMID: 21905200
- Elevated calcium levels induce CD43 capping, and macrophages remove the cells if their nucleolin receptors can bind to the poly-N-acetyllactosaminyl chains of capped CD43. PMID: 23400223
- Targeting CD43 in A549 lung cancer cells increased homotypic adhesion, decreased heterotypic adhesion and transendothelial migration, increased susceptibility to apoptosis, and increased vulnerability to lysis by NK cells. PMID: 23015282
- Despite high CD43 expression in both tumorous and nontumorous Langerhans cells (LCs), the JL1 epitope of CD43 is exposed in immature and neoplastic LCs. PMID: 22790855
- Compared to healthy controls, both CD43 mRNA and protein expressions were reduced in T cells from patients with SLE, and were inversely correlated with IgG. PMID: 22613599
- CD43 localizes to the nucleus, where it binds chromatin, co-localizes and co-immunoprecipitates with beta-catenin, and enhances the reporter gene expression regulated by beta-catenin. PMID: 22576689
- The capping of CD43 on the cell surface is a strong signal for phagocytosis, allowing phagocytes to differentiate between healthy and apoptotic cells without any additional membrane changes. PMID: 22466560
- The anti-adhesive function of CD43 in colon carcinoma cells plays a role in the tumorigenesis and metastasis of colorectal carcinoma cells. PMID: 22075155
- A negative feedback loop exists between p53 and CD43: CD43-dependent signaling activates p53, which in turn downregulates the expression of CD43. PMID: 21947346
- O-glycosylated CD43 and CD45 molecules on T cells regulate cell adhesion and favor the transmission of HTLV-1 from cell to cell. PMID: 22171268
- Data show that Pic, a class 2 SPATE protein produced by Shigella flexneri 2a, targets a broad range of human leukocyte glycoproteins, including CD43, CD44, CD45, CD93, CD162, and the surface-attached chemokine fractalkine. PMID: 21768350
- The tumor antigen UN1 has been identified as the transmembrane CD43 sialoglycoprotein. PMID: 21372249
- Expression of CD43 induces cell rounding, inhibition of cell re-attachment, augmentation of microvilli, and phosphorylation of Ezrin/Radixin/Moesin (ERM) in HEK293T cells. PMID: 21045567
- Engagement of CD43, presumably through the repressing transcription, initiates a Bad-dependent apoptotic pathway. PMID: 11773067
- CD43 can be considered a co-stimulatory cell surface constituent that modulates HIV-1 expression in T lymphocytes. PMID: 12045189
- Down-regulation of CD43 mRNA levels occurs during activation of the K562 cell line. This repression coincides with repression of the transcriptional activity of the CD43 gene promoter. PMID: 12411317
- This study examined the expression of CD43 in different human cell lines and tumor cell lines. PMID: 12499775
- While ezrin-associated CD43 protein is excluded from the inhibitory, i.e., noncytolytic, NK cell immune synapse (IS), it is homogeneously distributed across the IS of activating conjugates. PMID: 12626536
- Mucins from colon carcinoma patients contained MUC1-type mucins carrying both sialyl-Lewis a and x epitopes, and CD43-type sialyl-Lewis a mucins with only low levels of sialyl-Lewis x epitopes. PMID: 12820726
- Engagement of CD43 on normal human T lymphocytes as well as in Jurkat cells results in transient phosphorylation of the zeta-chain and enhanced association of ZAP-70 and Vav to the zeta-chain. PMID: 12902492
- Overexpression of SPN causes activation of the tumor suppressor proteins p53 and ARF 1. PMID: 14676827
- Four phage antibodies were isolated and used in a preliminary immunohistochemistry study of CD43 expression on frozen colorectal adenoma and carcinoma tissue. PMID: 14719063
- Findings suggest that the CD43 molecules expressed on CD4+ memory T cells may enhance costimulatory signaling, providing accessory functions to TCR-mediated activation processes. PMID: 15187099
- Microarray analysis of inflammatory genes reveals one group of genes coregulated by both stimuli and two further groups of target genes affected solely by costimulation or primarily by CD43. PMID: 15280197
- CD43 is expressed by a variety of carcinoma cell lines and plays a role in tumor cell-peritoneal adhesion, possibly through interactions with its putative ligand ICAM-1. PMID: 15449712
- Data suggest that PKCtheta plays a critical role in the co-stimulatory functions of CD43 in human T cells. PMID: 15522211
- CD43 is a T-cell E-selectin ligand distinct from PSGL-1, expanding its role in the regulation of T-cell trafficking. PMID: 16269612
- CD43 induces a signaling cascade that prolongs the duration of T cell receptor signaling, supporting the temporality with which certain molecules are engaged as a mechanism to fine tune T cell signal quality and ultimately immune function. PMID: 16751378
- Early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin. PMID: 16757688
- CD43 promotes cell growth and is a potential contributor to tumor development. PMID: 17891181
- Lymphoepithelial lesions pattern, CD43 coexpression, and clonal plasma cell component in extranodal marginal zone B-cell lymphoma (EMZL) are site-dependent, and the differences may aid in the diagnosis of EMZLs at different anatomic sites. PMID: 17979485
- Triggering CD43 and the underlying signaling pathways enhance LFA-1 adhesiveness, while CD43 also negatively regulates LFA-1 induction via other receptors by dynamically interacting with either LFA-1 or CD147. PMID: 17996943
- CD43 appears to be selectively expressed in a subset of adenoid cystic carcinomas, and its significance in salivary tumors is discussed. PMID: 18227725
- The cleavage of neutrophil leukosialin (CD43) by cathepsin G releases its extracellular domain and triggers its intramembrane proteolysis by presenilin/gamma-secretase. PMID: 18586676
- Streptococcus gordonii DL1 surface protein Hsa binds to the host cell membrane glycoproteins CD11b, CD43, and CD50. PMID: 18678668
Q&A
What is the SPN antigen and how does it relate to NuMA?
SPN antigen is a high molecular mass protein that relocates from the interphase nucleus to spindle poles during mitosis. Research has conclusively established that SPN and NuMA (Nuclear Mitotic Apparatus protein) are identical. Immunoprecipitated SPN antigen reacts with autoimmune human NuMA serum, and peptides derived from immunoprecipitated human SPN by cyanogen bromide cleavage show perfect alignment with sequences predicted for NuMA protein. This identity was confirmed through extensive protein sequence analysis covering more than fifty amino acids .
What roles does the SPN/NuMA protein play in cellular processes?
SPN/NuMA protein plays a critical functional role during mitosis, particularly in early mitotic stages. Experimental evidence indicates that it is essential for proper spindle formation and chromosome segregation. When SPN antibodies are introduced into cells during early mitotic phases (prophase, prometaphase, or metaphase), they significantly disrupt cell division, leading to defective cytokinesis or the formation of micronuclei in daughter cells . The protein appears less critical during later stages of mitosis, as antibody injection during anaphase produces fewer abnormalities, suggesting stage-specific requirements for SPN/NuMA function.
How are SPN monoclonal antibodies typically generated for research applications?
The generation of SPN monoclonal antibodies typically follows established hybridoma techniques similar to those used for other monoclonal antibodies. The process generally involves:
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Immunization of mice (often BALB/c strain) with the target protein
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Harvesting of B cells from immunized mice
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Fusion of B cells with myeloma cells to create hybridomas
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Screening of hybridomas for antibody production
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Cloning by limiting dilution to isolate monoclonal populations
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Characterization and verification of antibody specificity and function
While not specifically describing SPN antibody generation, related protocols describe immunizing mice subcutaneously with target proteins emulsified with Freund's adjuvant, followed by boosting with specific protein domains prior to hybridoma generation .
What methods are most effective for evaluating SPN monoclonal antibody specificity and functional activity?
Effective evaluation of SPN monoclonal antibodies should include multiple complementary techniques:
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Immunoprecipitation assays: To confirm binding to the target antigen (SPN/NuMA) and cross-reactivity with known related proteins
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Immunofluorescence microscopy: To verify proper localization patterns (nuclear in interphase, spindle poles during mitosis)
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Microinjection experiments: To assess functional effects on mitotic progression
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Western blotting: To confirm molecular weight and specificity
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Peptide mapping: Using techniques like cyanogen bromide cleavage to verify epitope recognition
The gold standard for functional validation involves microinjection into living cells during different cell cycle stages to observe phenotypic effects on mitosis and cytokinesis, as demonstrated in research with SPN-3 antibody .
How can microinjection of SPN antibodies be used to study mitotic mechanisms?
Microinjection of SPN/NuMA antibodies provides a powerful experimental approach for studying mitotic mechanisms by creating functional inhibition at specific cell cycle stages. The methodology includes:
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Timing-specific injections: Introducing antibodies at precise stages (prophase, prometaphase, metaphase, or anaphase) to determine stage-specific requirements
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Concentration optimization: Titrating antibody concentrations to achieve partial vs. complete inhibition
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Cell type selection: Using flat, adherent cells like PtK2 that allow clear visualization of mitotic stages
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Phenotypic analysis: Quantifying outcomes such as micronuclei formation, defective cytokinesis, and spindle abnormalities
Research has shown that injection of SPN-3 antibody during early mitotic stages (prophase, prometaphase, or metaphase) results in high frequencies of abnormal division (90%, 78%, and 77% respectively), while anaphase injection produces much lower abnormality rates (16%) . This approach allows precise temporal mapping of SPN/NuMA protein function during mitosis.
How do the effects of SPN monoclonal antibody microinjection compare to chemical mitotic inhibitors?
Studies have revealed surprising parallels between SPN-3 antibody microinjection and chemical mitotic inhibitors like colcemid and taxol. All three interventions can disrupt normal spindle formation and chromosome segregation, though through different mechanisms:
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SPN-3 antibody: Directly interferes with SPN/NuMA protein function at spindle poles
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Colcemid: Destabilizes microtubules, preventing proper spindle formation
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Taxol: Stabilizes microtubules, inhibiting normal dynamics required for chromosome movement
The similar phenotypic outcomes (micronuclei formation and cytokinesis defects) suggest that SPN/NuMA plays a critical role in the same cellular pathways affected by these chemical agents . This provides researchers with complementary tools to study mitotic regulation from different mechanistic perspectives.
What are the major challenges in generating highly specific SPN monoclonal antibodies?
Generating highly specific SPN monoclonal antibodies presents several challenges that researchers must address:
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Protein complexity: SPN/NuMA is a high molecular weight protein with multiple domains
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Cross-reactivity: Potential cross-reactivity with structurally similar nuclear proteins
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Conformational epitopes: Important functional epitopes may be conformational rather than linear
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Antibody class selection: Different isotypes (IgG vs. IgM) may have different functional properties in experimental applications
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Epitope accessibility: Some epitopes may be masked in native protein complexes
Successful antibody development requires careful immunization strategies, thorough screening methods, and validation in multiple assay formats to ensure both specificity and functional activity.
How can researchers verify that observed phenotypes are specifically due to SPN/NuMA inhibition rather than off-target effects?
Verifying specificity of observed phenotypes requires multiple control experiments:
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Control antibodies: Using isotype-matched non-specific antibodies for injection
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Rescue experiments: Co-injecting purified SPN/NuMA protein with the antibody to neutralize its effects
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Multiple antibody validation: Testing different monoclonal antibodies targeting distinct epitopes of SPN/NuMA
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Genetic approaches: Comparing antibody effects with genetic knockdown/knockout phenotypes
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Dose-response relationships: Establishing clear correlations between antibody concentration and phenotypic severity
Additionally, careful comparisons with known mitotic inhibitors like colcemid and taxol can help differentiate specific SPN/NuMA-related effects from general mitotic disruption .
How are SPN monoclonal antibodies being applied in cancer research?
SPN monoclonal antibodies have potential applications in cancer research based on the critical role of SPN/NuMA in mitosis:
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Cell division markers: As tools to study aberrant mitotic processes in cancer cells
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Therapeutic exploration: Investigating whether targeting SPN/NuMA could inhibit cancer cell proliferation
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Biomarker development: Assessing whether SPN/NuMA expression or localization correlates with cancer aggressiveness or treatment response
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Mechanistic studies: Understanding how cancer-associated mutations might affect SPN/NuMA function during cell division
While direct cancer applications of SPN antibodies are still emerging, the established role of SPN/NuMA in mitotic regulation suggests potential relevance to understanding and targeting cancer cell proliferation.
What methodological approaches can be used to study the interaction between SPN/NuMA and other mitotic proteins?
Several methodological approaches are valuable for studying SPN/NuMA interactions with other mitotic proteins:
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Co-immunoprecipitation: Pulling down SPN/NuMA and identifying binding partners
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Proximity labeling: Using BioID or APEX techniques to identify proteins in close proximity during mitosis
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Fluorescence microscopy: Co-localization studies with other mitotic components
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FRET/BRET analysis: Measuring direct protein-protein interactions in living cells
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Cross-linking mass spectrometry: Identifying interaction interfaces between SPN/NuMA and binding partners
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In vitro binding assays: Using purified components to assess direct interactions
These approaches can reveal how SPN/NuMA coordinates with other proteins to regulate spindle formation and chromosome segregation during mitosis.
What cell types are most suitable for studying SPN/NuMA function using monoclonal antibodies?
The selection of appropriate cell types is critical for successful studies of SPN/NuMA function:
| Cell Type | Advantages | Limitations | Best Applications |
|---|---|---|---|
| PtK2 (rat kangaroo) | Flat morphology, clear visualization of mitotic structures | Non-human origin | Microinjection, live imaging |
| HeLa | Human origin, well-characterized | Cancer-derived, potential abnormal mitotic regulation | Biochemical studies, fixed-cell imaging |
| RPE-1 | Non-transformed human cells, normal karyotype | More challenging for microinjection | Physiologically relevant studies |
| U2OS | Large size, adherent | Cancer-derived | High-resolution imaging |
Research with SPN-3 antibody has successfully used PtK2 cells for microinjection experiments, allowing clear visualization of mitotic defects following antibody introduction .
What controls should be included when conducting functional studies with SPN monoclonal antibodies?
Rigorous experimental design for SPN antibody studies should include:
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Isotype controls: Non-specific antibodies of the same isotype and concentration
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Temporal controls: Injections at different cell cycle stages to establish stage-specific effects
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Concentration series: Multiple antibody concentrations to establish dose-dependent responses
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Cell type controls: Testing in multiple cell lines to ensure generalizability of findings
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Comparison standards: Reference compounds like colcemid or taxol with known mitotic effects
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Blocking controls: Pre-incubation of antibodies with purified antigen to neutralize specific binding
These controls help distinguish specific effects of SPN/NuMA inhibition from non-specific effects of antibody introduction or experimental manipulation.
How might new antibody engineering technologies enhance SPN/NuMA research?
Emerging antibody technologies offer exciting opportunities for advancing SPN/NuMA research:
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Single-domain antibodies: Smaller antibody fragments that may access epitopes unavailable to conventional antibodies
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Intrabodies: Engineered antibodies that function within living cells without microinjection
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Bispecific antibodies: Targeting SPN/NuMA along with interaction partners simultaneously
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Optogenetic antibody systems: Light-controlled antibody activity for precise temporal control
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Degradation-targeting antibodies: Inducing selective degradation of SPN/NuMA at specific cell cycle stages
These approaches could enable more precise spatial and temporal control over SPN/NuMA function, revealing new aspects of its role in mitotic regulation.
What are the potential applications of combining SPN monoclonal antibodies with advanced imaging technologies?
Integration of SPN antibodies with cutting-edge imaging offers powerful new research possibilities:
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Super-resolution microscopy: Nanoscale visualization of SPN/NuMA localization and dynamics
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Live-cell CLEM (Correlative Light and Electron Microscopy): Connecting functional perturbations with ultrastructural changes
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Lattice light-sheet microscopy: Long-term 3D imaging of mitotic progression following antibody introduction
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Single-molecule tracking: Following individual SPN/NuMA molecules during spindle assembly
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Expansion microscopy: Physical enlargement of cellular structures for enhanced resolution of SPN/NuMA distribution
These combined approaches could reveal previously undetectable aspects of SPN/NuMA function in organizing mitotic structures and regulating chromosome segregation.
