NUMA1 Recombinant Monoclonal Antibody

What is NUMA1 and why is it significant in mitotic research?

NUMA1 (Nuclear Mitotic Apparatus Protein 1), also known as NMP22 or Nuclear matrix protein-22, is a critical component involved in nuclear organization and spindle pole formation during mitosis. It plays essential roles in maintaining nuclear structural integrity during interphase and reorganizing the nucleus during mitotic division. The protein's importance in cell division makes it a valuable target for cell cycle research, cancer studies, and investigations into chromosome segregation mechanisms .

NUMA1 antibodies are particularly useful for studying mitotic spindle assembly, nuclear reformation, and the spatial organization of chromatin. The protein's distribution changes dramatically during the cell cycle, making it an excellent marker for cell cycle progression studies.

What are the key differences between traditional and recombinant monoclonal antibodies for NUMA1?

Traditional monoclonal antibodies for NUMA1 are typically generated in animals (often mice), which presents several limitations compared to recombinant alternatives:

Recombinant monoclonal antibodies offer significant advantages in terms of reproducibility and standardization, which are critical issues in research. As noted in research on antibody development, "traditional antibodies generated in animals" face problems with "lack of standardization leading to problems with reproducibility," while recombinant antibodies provide "low-cost, high-yield preparations" with consistent properties .

What applications are NUMA1 monoclonal antibodies validated for?

NUMA1 monoclonal antibodies have been validated for multiple applications, with specific dilution recommendations varying by manufacturer and application:

It's important to note that "optimal working dilutions must be determined by end user" as indicated in multiple sources, as the optimal conditions may vary depending on specific experimental conditions and sample types .

How should researchers approach validation of epitope specificity for NUMA1 recombinant monoclonal antibodies?

Proper validation of epitope specificity is crucial for ensuring reliable results with NUMA1 antibodies. A comprehensive validation approach should include:

• Immunogen verification: Confirm the specific sequence used as immunogen. For example, one NUMA1 antibody uses "a synthesized peptide derived from human NuMA" as its immunogen, while another uses "NUMA1 (NP_006176, 200 a.a. ~ 306 a.a) partial recombinant protein" . Understanding the exact epitope helps predict potential cross-reactivity.

• Western blot with recombinant controls: Run parallel blots with recombinant NUMA1 protein as a positive control. The calculated molecular weight of NUMA1 is approximately 42,051 Da, though the observed molecular weight is often around 66 kDa due to post-translational modifications .

• Knockout/knockdown validation: Use NUMA1 knockout or knockdown cell lines as negative controls to confirm antibody specificity.

• Cross-reactivity assessment: Test the antibody against samples from multiple species to verify the claimed species reactivity. Most NUMA1 antibodies react with human samples, and some also detect mouse, rat, and porcine NUMA1 .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to block specific binding sites before application to samples.

• Orthogonal method comparison: Compare results with alternative detection methods or antibodies targeting different NUMA1 epitopes.

The validation should be systematically documented, including positive and negative controls for each application the antibody will be used for.

What are the critical factors for optimizing immunofluorescence experiments with NUMA1 antibodies?

When designing immunofluorescence experiments with NUMA1 antibodies, researchers should consider:

• Fixation method: NUMA1 detection can be sensitive to fixation methods. Paraformaldehyde (4%) is commonly used, but methanol fixation may better preserve nuclear structure for NUMA1 visualization.

• Permeabilization optimization: Since NUMA1 is a nuclear protein, adequate permeabilization is essential. Test different detergents (Triton X-100, saponin) and concentrations to optimize access to nuclear epitopes without disrupting structure.

• Blocking considerations: Use species-appropriate blocking reagents to minimize background signal. BSA (0.4-0.5 mg/ml) is often included in antibody formulations and can be effective for blocking .

• Signal amplification strategies: For low-abundance epitopes or weak signals, consider using secondary antibody amplification systems or tyramide signal amplification.

• Co-localization studies: NUMA1 distribution changes dramatically during mitosis, so co-staining with cell cycle markers can provide valuable context. When designing multi-color experiments, ensure spectral separation between fluorophores.

• Dilution optimization: Starting with the manufacturer's recommended dilution (typically 5-20 μg/mL for ICC applications), perform a dilution series to determine optimal antibody concentration for your specific samples .

• Imaging parameters: Due to NUMA1's nuclear localization and redistribution during mitosis, confocal microscopy with Z-stacking is often necessary to properly visualize its three-dimensional distribution.

• Quantification approaches: For quantitative analysis of NUMA1 distribution, establish consistent imaging and analysis parameters, with appropriate controls for normalization.

How can researchers modify NUMA1 recombinant monoclonal antibodies for specialized applications?

The recombinant nature of these antibodies provides multiple opportunities for customization to meet specific research needs:

• Species specificity customization: The primary sequence of recombinant antibodies can be modified to enhance cross-reactivity with NUMA1 from different species or to make them species-specific, addressing the variability in reactivity observed across commercial antibodies .

• Format conversion: Researchers can generate different antibody formats from the same binding domain:

  • Convert to single-chain variable fragments (scFv) for improved tissue penetration

  • Generate Fab fragments for applications requiring monovalent binding

  • Create full-length, bivalent antibodies from scFv for enhanced avidity

• Fusion protein creation: The antibody coding sequence can be fused with:

  • Fluorescent proteins for direct visualization without secondary antibodies

  • Enzymes like HRP or AP for direct detection in blotting or IHC

  • Affinity tags (His, GST, etc.) for simplified purification and detection

• Isotype switching: Converting between different isotypes (e.g., IgG1, IgG2a, IgG2b) can optimize the antibody for specific applications like complement activation or Fc receptor binding .

• Affinity maturation: Directed evolution approaches can enhance binding affinity or specificity through techniques like phage display combined with stringent selection.

These modifications require molecular biology expertise but provide powerful customization options that are impossible with traditional hybridoma-derived antibodies.

What troubleshooting approaches should be used when NUMA1 antibodies yield unexpected results?

When facing challenges with NUMA1 antibody experiments, consider these methodical troubleshooting approaches:

• Non-specific binding in Western blots:

  • Increase blocking stringency (5% milk or BSA)

  • Optimize primary antibody concentration (test range from 0.01-2 μg/mL)

  • Increase wash duration and frequency

  • Try alternative membrane types

  • Use gradient gels to better resolve the 66 kDa NUMA1 band from nearby proteins

• Weak or no signal in immunofluorescence:

  • Verify epitope accessibility with different fixation methods

  • Ensure adequate permeabilization for nuclear protein detection

  • Try antigen retrieval methods (if using fixed tissue)

  • Increase antibody concentration within recommended range (5-20 μg/mL)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Check for potential epitope masking during cell cycle phases

• Inconsistent immunoprecipitation results:

  • Optimize lysis conditions to maintain NUMA1 native conformation

  • Test different IP protocols (direct vs. indirect coupling)

  • Increase starting material amount

  • Verify antibody compatibility with IP buffer components

  • Confirm that the antibody recognizes the native (non-denatured) form of NUMA1

• Batch-to-batch variation:

  • Use recombinant NUMA1 antibodies for greater consistency

  • Establish internal validation procedures for each new lot

  • Maintain consistent experimental conditions

  • Include positive control samples in each experiment

• Cross-reactivity concerns:

  • Validate with knockout/knockdown controls

  • Perform peptide competition assays

  • Compare results with antibodies targeting different NUMA1 epitopes

Detailed documentation of all experimental parameters and systematic variation of one factor at a time will facilitate efficient troubleshooting.

What are the optimal storage and handling conditions for NUMA1 recombinant monoclonal antibodies?

Proper storage and handling are crucial for maintaining antibody activity and experimental reproducibility:

"Store at -20°C for one year. For short term storage and frequent use, store at 4°C for up to one month. Avoid repeated freeze-thaw cycles" . This guidance is consistent across multiple sources, emphasizing the importance of minimizing freeze-thaw cycles to maintain antibody integrity.

How do different fixation and sample preparation methods affect NUMA1 detection?

Sample preparation significantly impacts NUMA1 detection, with different methods offering advantages for specific applications:

Different applications may require specific optimization of these parameters, and preliminary testing with positive control samples is recommended before proceeding with experimental samples.

What controls are essential when working with NUMA1 antibodies in various applications?

Implementing appropriate controls is crucial for ensuring valid and interpretable results:

• Essential positive controls:

  • Cell lines known to express NUMA1 (e.g., K-562 cells have been validated for Western blot)

  • Recombinant NUMA1 protein (full-length or the specific domain targeted by the antibody)

  • Tissues with known NUMA1 expression (e.g., human kidney tissue has been validated for IHC)

• Negative controls:

  • Isotype-matched control antibodies at the same concentration

  • Secondary antibody-only controls to assess background

  • NUMA1 knockout or knockdown samples where available

  • Pre-absorption with immunizing peptide to confirm specificity

• Controls specific to application:

  • Western blot: Loading controls (β-actin, GAPDH); molecular weight markers

  • IHC/ICC: Adjacent serial sections with isotype control; biological positive and negative tissue controls

  • IP: Input sample control; IgG control IP; reverse IP when possible

  • IF: Cell cycle stage controls (NUMA1 distribution varies during mitosis)

• Quantification controls:

  • Standard curves with recombinant protein for quantitative applications

  • Internal reference standards for comparing across experiments

  • Technical replicates to assess method reproducibility

• Validation across applications:

  • Confirm findings with orthogonal methods when possible

  • Use multiple antibodies targeting different NUMA1 epitopes

  • Compare results across multiple cell lines or tissue types

Documentation of all controls is essential for publication and ensuring experimental reproducibility.

How can researchers generate customized NUMA1 antibody fragments for specialized applications?

Researchers can produce customized NUMA1 antibody fragments following established recombinant antibody engineering approaches:

• Sequence determination:

  • For existing hybridoma cell lines, obtain mRNA transcriptome and generate cDNA library

  • Identify antibody sequences through whole transcriptome shotgun sequencing

  • Classify antibody isotype (e.g., "IgG2b with a kappa LC" or "Ig")

• Fragment generation methods:

  • Fab fragments: Enzymatic digestion with papain followed by protein A purification

  • F(ab')2 fragments: Pepsin digestion removing the Fc portion while maintaining bivalency

  • scFv construction: PCR amplification of VH and VL domains with flexible linker insertion

  • Nanobodies: Based on camelid single-domain antibodies for minimal size

• Expression systems:

  • Bacterial (E. coli): Cost-effective but lacks mammalian glycosylation

  • Mammalian cells (CHO, HEK293): Provides proper folding and post-translational modifications

  • Insect cells: Intermediate between bacterial and mammalian systems

  • Cell-free systems: Rapid production for screening purposes

• Functional modifications:

  • Add affinity tags for purification (His, GST, FLAG)

  • Incorporate site-specific biotinylation sequences

  • Engineer cysteine residues for site-specific conjugation

  • Add fluorescent protein fusions for direct detection

• Purification approaches:

  • Affinity chromatography (Protein A/G for full antibodies)

  • Immobilized metal affinity chromatography for His-tagged fragments

  • Size exclusion chromatography for final polishing

  • Endotoxin removal for cell-based applications

This process allows "customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies" , providing researchers with highly tailored tools for specialized applications.

How are NUMA1 antibodies being used in current mitosis and cell division research?

NUMA1 antibodies are important tools in several active research areas:

• Mitotic spindle assembly studies:

  • Tracking NUMA1 redistribution from the nucleus to spindle poles during mitosis

  • Investigating interactions with dynein and other motor proteins

  • Studying the role of NUMA1 in microtubule organization and stability

  • Examining NUMA1 phosphorylation in regulating spindle function

• Cell cycle checkpoint research:

  • Analyzing NUMA1's relationship with checkpoint proteins like BubR1 and Mad2

  • Investigating NUMA1's role in sensing tension across the mitotic spindle

  • Studying NUMA1 in chromosome alignment and segregation mechanisms

• Cancer research applications:

  • Using NUMA1 as a marker for proliferating cells in tumor samples

  • Examining NUMA1 expression levels in cancer progression

  • Investigating NUMA1 alterations in chromosomally unstable cancers

  • Studying NUMA1 as a potential therapeutic target

• Nuclear architecture studies:

  • Examining NUMA1's role in maintaining nuclear structural integrity

  • Investigating interactions with lamins and other nuclear envelope proteins

  • Studying NUMA1's function in chromosome territory organization

  • Analyzing nuclear reformation after mitosis

NUMA1 antibodies are particularly valuable in studies examining the "kinetochore-associated and spindle assembly checkpoint" mechanisms, which are critical for proper cell division and genomic stability .

What are the emerging technologies for enhanced detection and analysis using NUMA1 antibodies?

Several innovative approaches are expanding the utility of NUMA1 antibodies:

• Super-resolution microscopy applications:

  • STORM and PALM imaging for nanoscale localization of NUMA1

  • SIM for improved visualization of NUMA1 distribution in the nuclear matrix

  • Expansion microscopy for physical magnification of NUMA1 structures

• Live-cell imaging strategies:

  • Mini-antibody formats with improved cell penetration

  • Antibody fragment-fluorescent protein fusions

  • SNAP/CLIP-tag labeling systems for pulse-chase experiments

  • nanobody-based detection for reduced interference with native functions

• Multiplexed detection methods:

  • Mass cytometry (CyTOF) incorporating NUMA1 with other cell cycle markers

  • Cyclic immunofluorescence for sequential antibody staining

  • DNA-barcoded antibodies for spatial transcriptomics integration

  • Highly multiplexed tissue imaging using spectral deconvolution

• Single-cell analysis integration:

  • Combining NUMA1 antibody detection with single-cell RNA sequencing

  • Correlative light and electron microscopy for ultrastructural context

  • Spatial proteomics approaches integrating NUMA1 localization data

• High-content screening applications:

  • Automated image analysis of NUMA1 distribution phenotypes

  • Machine learning classification of mitotic abnormalities

  • Drug screening using NUMA1 localization as a readout

These technologies benefit from the "practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies" that provide consistent performance across experiments .

What considerations are important when selecting between different formats of NUMA1 antibodies?

Researchers should consider these factors when selecting the appropriate NUMA1 antibody format:

When making this selection, researchers should consider:

• Application requirements: Different formats perform optimally in specific applications. For example, full-length IgGs are often preferred for Western blotting, while smaller fragments may be advantageous for tissue penetration in IHC.

• Expression system impact: The choice of production system affects glycosylation and folding. Mammalian expression systems generally provide properly folded and post-translationally modified antibodies.

• Clonality considerations: Monoclonal antibodies offer consistency and specificity, while polyclonal preparations may provide signal amplification by recognizing multiple epitopes.

• Species compatibility: For multi-color immunofluorescence, selecting antibodies from different host species can simplify detection strategies.

• Storage stability: Different formats have varying stability profiles. Full-length IgGs tend to be most stable during storage, while some antibody fragments may require special stabilization measures.

Converting between formats is increasingly feasible with recombinant technology, allowing researchers to select the optimal format for each specific application.

What are the most common technical challenges with NUMA1 antibodies and how can they be resolved?

Researchers frequently encounter these challenges when working with NUMA1 antibodies:

• Nuclear localization detection issues:

  • Problem: Inadequate nuclear staining despite confirmed NUMA1 expression

  • Solutions:

    • Optimize permeabilization (try 0.5% Triton X-100 for 10-15 minutes)

    • Test different fixation methods (compare PFA vs. methanol)

    • Ensure adequate antibody concentration (5-20 μg/mL for IHC/ICC)

    • Consider antigen retrieval for fixed samples

• Cell cycle-dependent detection variability:

  • Problem: Inconsistent NUMA1 staining patterns across samples

  • Solutions:

    • Synchronize cells to control for cell cycle stage

    • Co-stain with cell cycle markers to contextualize NUMA1 distribution

    • Document specific patterns associated with different mitotic phases

    • Use positive control cells with known NUMA1 distribution patterns

• Western blot molecular weight discrepancies:

  • Problem: Observed band at 66 kDa vs. calculated 42 kDa molecular weight

  • Solutions:

    • Run gradient gels for better resolution

    • Include positive control recombinant NUMA1

    • Verify with alternative NUMA1 antibodies

    • Consider post-translational modifications affecting migration

• Cross-reactivity concerns:

  • Problem: Unexpected bands or staining patterns

  • Solutions:

    • Perform peptide competition assays

    • Test in knockout/knockdown systems

    • Optimize blocking conditions (5% BSA or milk)

    • Increase washing stringency and duration

• Low signal-to-noise ratio:

  • Problem: High background obscuring specific signal

  • Solutions:

    • Titrate antibody to optimal concentration

    • Increase blocking time and concentration

    • Use more stringent washing (longer, more frequent)

    • Try alternative secondary antibodies

    • Consider specialized blocking reagents (e.g., for endogenous biotin)

Methodical approach to troubleshooting, changing one variable at a time and maintaining detailed records of conditions tested, will facilitate efficient resolution of these common challenges.

How should researchers interpret and validate unexpected NUMA1 staining patterns?

When faced with unexpected NUMA1 staining patterns, follow this systematic validation approach:

• Pattern characterization:

  • Document precise localization (nuclear, cytoplasmic, punctate, diffuse)

  • Compare with published NUMA1 distribution patterns

  • Quantify the frequency of unusual patterns across multiple samples

  • Correlate patterns with cell cycle stages, cell types, or treatments

• Technical validation:

  • Repeat with alternative fixation and permeabilization methods

  • Test with antibodies targeting different NUMA1 epitopes

  • Perform co-localization studies with known NUMA1 interactors

  • Compare results across multiple detection methods (IF, IHC, WB)

• Biological validation:

  • Correlate patterns with functional assays

  • Test in NUMA1 knockdown/knockout systems

  • Examine effects of cell cycle perturbations

  • Investigate under different physiological stresses

• Advanced validation approaches:

  • Perform subcellular fractionation followed by Western blotting

  • Use proximity ligation assays to confirm suspected interactions

  • Employ CRISPR-tagged endogenous NUMA1 as a reference

  • Consider mass spectrometry validation of unexpected NUMA1 locations

• Potential biological interpretations:

  • Post-translational modifications affecting localization

  • Alternative splice variants with different distribution patterns

  • Cell type-specific NUMA1 functions

  • Disease-associated mislocalization

Remember that unexpected findings may represent novel biology rather than technical artifacts, particularly when they are reproducible across multiple experimental approaches and validated with appropriate controls.

What quality control measures should researchers implement when working with newly received NUMA1 antibodies?

Before using a new NUMA1 antibody in critical experiments, perform these quality control checks:

• Initial physical inspection:

  • Verify absence of precipitation or cloudiness

  • Check for appropriate volume and appearance

  • Confirm proper storage conditions during shipping

  • Record lot number and expiration date

• Documentation review:

  • Check the immunogen sequence to understand the specific epitope

  • Review validated applications and recommended conditions

  • Note any specific warnings or limitations

  • Verify species reactivity claims against your experimental model

• Basic validation tests:

  • Western blot: Run against positive control lysates (e.g., K-562 cells)

  • Immunofluorescence: Test on fixed cells with known NUMA1 expression

  • Dilution series: Determine optimal working concentration for each application

  • Blocking test: Compare specific vs. non-specific binding

• Advanced validation (as needed):

  • Test with recombinant NUMA1 protein

  • Compare against previously validated NUMA1 antibodies

  • Perform peptide competition assays

  • Validate in knockout/knockdown systems if available

• Long-term quality monitoring:

  • Aliquot to avoid freeze-thaw cycles

  • Include consistent positive controls in all experiments

  • Maintain detailed records of performance over time

  • Test new lots against previous lots before switching

The thermal stability of NUMA1 antibodies has been tested by "accelerated thermal degradation test, that is, incubate the protein at 37°C for 48h," with acceptable stability defined as "loss rate is less than 5% within the expiration date under appropriate storage condition" . Similar stability testing can be performed in-house for critical applications.

How might recombinant NUMA1 antibody technology evolve to address current research limitations?

Several emerging trends are likely to expand the capabilities of NUMA1 antibody research:

• Enhanced recombinant production platforms:

  • Development of standardized expression systems with improved yield and consistency

  • Implementation of automated antibody production pipelines

  • Creation of open-source antibody sequence repositories

  • Adoption of artificial intelligence for antibody design optimization

• Advanced engineering for improved functionality:

  • Site-specific conjugation for precise labeling

  • Stimulus-responsive antibodies with conditional binding properties

  • Multivalent constructs targeting NUMA1 plus interacting partners

  • pH or redox-sensitive formats for environment-specific activation

• Intracellular antibody applications:

  • Improved cell-penetrating antibody formats

  • Genetically encoded intrabodies for real-time NUMA1 monitoring

  • Targeted protein degradation using antibody-degrader conjugates

  • Antibody-mediated manipulation of NUMA1 function

• Integration with emerging technologies:

  • Cryo-electron tomography compatible antibody probes

  • Quantum dot conjugates for long-term tracking

  • Expansion microscopy-optimized antibody formats

  • Integration with spatial multi-omics platforms

These advances will address current limitations in antibody research, moving toward the goal of generating "low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences" , ultimately providing more reliable and versatile tools for NUMA1 research.

What novel applications might emerge from combining NUMA1 antibodies with advanced imaging technologies?

The integration of NUMA1 antibodies with cutting-edge imaging approaches will enable new research directions:

• 4D imaging of NUMA1 dynamics:

  • Real-time tracking of NUMA1 redistribution throughout the cell cycle

  • Quantitative analysis of NUMA1 movement rates and trajectories

  • Correlation of NUMA1 dynamics with force generation during mitosis

  • Multi-day live imaging of NUMA1 across multiple cell divisions

• Nanoscale structural insights:

  • Super-resolution microscopy revealing NUMA1 organization at spindle poles

  • Examination of NUMA1's role in organizing nuclear architecture at molecular scale

  • Visualization of NUMA1-microtubule interactions during spindle assembly

  • Quantification of NUMA1 clustering and oligomerization states

• Correlative multi-modal imaging:

  • CLEM (Correlative Light and Electron Microscopy) to place NUMA1 in ultrastructural context

  • Correlative super-resolution and atomic force microscopy for structural-mechanical insights

  • Integration of NUMA1 imaging with genomic visualization techniques

  • Multi-scale imaging from tissue to molecular level

• Functional imaging approaches:

  • FRET/FLIM studies of NUMA1 protein-protein interactions

  • Optogenetic manipulation of NUMA1 with simultaneous imaging

  • Force measurement during NUMA1-mediated spindle positioning

  • Tracking consequences of NUMA1 perturbation in real-time

These applications will benefit from the ability to "customize" antibody properties, "including species specificity" and generating specialized antibody fragments optimized for specific imaging modalities .