mzt2 Antibody

What is MZT2A and why is it significant in biological research?

MZT2A (also known as GCP8a or FAM128A) is a protein that mediates cell septation signaling during growth by regulating microtubule polymerization and depolymerization at the centrosome. Initially identified in 2010 by Hutchins et al. as MOZART2, it was further characterized by Teixido-Travesa et al. as a component of the γ-tubulin ring complex (γ-TuRC) with a size of approximately 20 kDa . Its significance stems from its crucial role in microtubule nucleation activity, with research showing that insufficient MZT2A expression impairs this process and reduces cell proliferation rates . Recent studies have also linked MZT2A to cancer progression, particularly in non-small-cell lung cancer (NSCLC), where its overexpression correlates with poor patient prognosis .

What types of MZT2A antibodies are available for research applications?

Based on current research sources, there are several types of MZT2A antibodies available:

For monoclonal antibodies, several have been developed against human MZT2A using purified His-tagged recombinant human MZT2A . For polyclonal antibodies, some are raised against specific immunogens designed to target particular epitopes of MZT2A . Both types offer complementary advantages: monoclonals provide high specificity for particular epitopes, while polyclonals recognize multiple epitopes, potentially offering greater detection sensitivity under certain experimental conditions.

How should MZT2A antibodies be validated before experimental use?

Methodological validation of MZT2A antibodies should follow a multi-step approach:

• Western blot analysis: Validate using positive control samples known to express MZT2A (e.g., MCF-7 cell lysates). Look for specific band detection at the expected molecular weight of approximately 20 kDa .

• Immunohistochemistry controls: Include both positive tissue controls (e.g., human brain tissue) and negative controls (primary antibody omission or isotype controls) .

• Cross-reactivity assessment: Test against samples from multiple species if cross-species reactivity is claimed. Documented reactivity includes human, mouse, rat, and bovine samples .

• Knockout/knockdown validation: The gold standard involves comparing antibody signal between wild-type samples and those where MZT2A has been knocked down using siRNA or CRISPR-Cas9 technologies .

• Flow cytometry validation: For antibodies intended for flow cytometry applications, compare staining patterns against matched isotype controls and unstimulated versus stimulated samples using appropriate positive control cell types .

What are the optimal conditions for using MZT2A antibodies in Western blotting?

For optimal Western blotting with MZT2A antibodies, the following methodological approach is recommended:

• Sample preparation: Extract proteins from target cells (e.g., MCF-7 cells or NSCLC cell lines) using standard lysis buffers containing protease inhibitors. Load 15-20 μg protein per lane for cell lines with expected MZT2A expression .

• Gel electrophoresis: Use 10-12% SDS-PAGE gels to achieve optimal separation of the ~20 kDa MZT2A protein.

• Transfer conditions: Semi-dry or wet transfer to PVDF membranes at 100V for 60-90 minutes in standard transfer buffer.

• Blocking: Block membranes for 1 hour at room temperature using 5% non-fat milk or BSA in TBST.

• Primary antibody dilution: For monoclonal antibodies, a dilution range of 1:5,000-1:10,000 is recommended. Incubate overnight at 4°C .

• Secondary antibody: Use HRP-conjugated secondary antibodies at 1:5,000-1:10,000 dilution, incubating for 1 hour at room temperature.

• Detection: Develop using ECL substrate, with expected band detection at approximately 20 kDa.

• Controls: Include positive control lysates from cells known to express MZT2A and negative controls (e.g., MZT2A-knockdown samples) to confirm specificity.

How can MZT2A antibodies be effectively utilized in immunohistochemistry protocols?

For successful immunohistochemistry with MZT2A antibodies, follow this methodological workflow:

• Tissue preparation: For formalin-fixed paraffin-embedded (FFPE) tissues, section at 4-6 μm thickness. For frozen sections, prepare 8-10 μm thick sections .

• Antigen retrieval: For FFPE tissues, heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended. Maintain at 95-98°C for 15-20 minutes, then cool to room temperature.

• Peroxidase and protein blocking: Block endogenous peroxidase using 3% H₂O₂ for 10 minutes, followed by protein blocking with 2-5% normal serum.

• Antibody dilution and incubation: For MZT2A monoclonal antibodies, dilute 1:1,000-1:5,000 in blocking buffer and incubate at 4°C overnight .

• Secondary detection: Apply biotinylated secondary antibody followed by peroxidase-conjugated streptavidin-biotin complex. For fluorescence detection, use appropriate fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488) .

• Visualization: For chromogenic detection, develop with DAB and counterstain with hematoxylin. For fluorescence, counterstain nuclei with DAPI .

• Controls: Include positive control tissues (e.g., human brain tissue) where MZT2A expression has been confirmed. Include negative controls by omitting primary antibody .

What are the key considerations for immunofluorescence experiments using MZT2A antibodies?

For immunofluorescence applications with MZT2A antibodies, researchers should consider these methodological details:

• Cell preparation: Culture cells on glass coverslips or chamber slides. Fix with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilization: Permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes to allow antibody access to intracellular MZT2A.

• Blocking: Block with 1-5% BSA or normal serum for 30-60 minutes at room temperature.

• Primary antibody incubation: Dilute MZT2A antibody 1:1,000-1:5,000 and incubate overnight at 4°C in a humidified chamber .

• Secondary antibody: Use fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488-conjugated anti-mouse IgG for mouse monoclonal antibodies) at manufacturer-recommended dilutions, typically 1:200-1:1,000 .

• Nuclear counterstaining: Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes.

• Mounting and imaging: Mount using anti-fade mounting medium and image using confocal or fluorescence microscopy.

• Expected localization: Look for MZT2A localization primarily at the centrosome, consistent with its role in microtubule organization .

What strategies can address weak or absent MZT2A antibody signals in Western blots?

When encountering weak or absent MZT2A signals in Western blots, consider these methodological interventions:

• Protein extraction optimization: MZT2A is associated with the centrosome, which can be difficult to extract with standard lysis buffers. Use RIPA buffer supplemented with 1% NP-40 and brief sonication to enhance extraction of centrosomal proteins.

• Sample concentration: If signal is weak, consider using immunoprecipitation to concentrate MZT2A before Western blotting.

• Transfer efficiency: For small proteins like MZT2A (~20 kDa), use methanol-containing transfer buffers and modify transfer conditions (lower voltage for longer time) to prevent protein loss.

• Antibody optimization: If standard dilutions (1:5,000-1:10,000) provide weak signals, test more concentrated antibody solutions (1:1,000-1:2,000) .

• Signal enhancement: Use high-sensitivity ECL substrates or signal amplification systems for low-abundance proteins.

• Membrane selection: PVDF membranes may provide better protein retention than nitrocellulose for small proteins like MZT2A.

• Expression verification: Confirm MZT2A expression in your samples using RT-PCR, as expression levels vary significantly between tissue types and cell lines .

How can researchers address cross-reactivity issues with MZT2A antibodies?

When experiencing cross-reactivity problems with MZT2A antibodies, implement these methodological solutions:

• Epitope analysis: Review antibody documentation for the specific epitope recognized. Choose antibodies targeting unique regions of MZT2A to minimize cross-reactivity with related proteins.

• Blocking optimization: Increase blocking stringency by using 5% BSA instead of milk, or add 0.1-0.3% Tween-20 to reduce non-specific binding.

• Validation controls: Include MZT2A knockdown or knockout samples as negative controls to distinguish specific from non-specific signals.

• Wash stringency: Increase wash duration and number of washes with TBST buffer to remove non-specifically bound antibodies.

• Competitive blocking: Pre-incubate the antibody with recombinant MZT2A protein before application to verify signal specificity.

• Affinity purification: For polyclonal antibodies, consider affinity purification against the immunizing peptide to enrich for specific antibodies.

• Secondary antibody control: Include a control omitting primary antibody to identify potential secondary antibody cross-reactivity.

What are the critical parameters for storing and handling MZT2A antibodies to maintain reactivity?

To preserve MZT2A antibody performance over time, observe these methodological storage and handling guidelines:

• Storage temperature:

  • Short-term (up to 1 month): 2-8°C for both lyophilized and reconstituted antibodies

  • Long-term: -20°C for reconstituted antibodies

• Reconstitution protocol: For lyophilized antibodies, reconstitute with 100 μL sterile-filtered ultrapure water to achieve a 1 mg/mL concentration. Centrifuge briefly to remove any insoluble material .

• Aliquoting: After reconstitution, prepare small aliquots (10-20 μL) to minimize freeze-thaw cycles, which can degrade antibody performance.

• Freeze-thaw limitation: Limit to ≤5 freeze-thaw cycles to maintain antibody activity.

• Working solution preparation: Dilute antibodies in fresh buffer immediately before use rather than storing diluted solutions.

• Preservatives: Addition of 0.02-0.05% sodium azide is recommended for solutions stored at 2-8°C, but ensure this is compatible with your downstream applications.

• Expiration tracking: Document reconstitution date and discard antibodies 12 months after reconstitution, even if stored properly .

How can MZT2A antibodies be employed to investigate its role in cancer progression?

MZT2A has emerging significance in cancer biology, particularly in NSCLC. Here's a methodological approach for investigating its role using antibodies:

• Expression profiling: Use validated MZT2A antibodies for immunohistochemical staining of tumor microarrays containing matched tumor and normal tissue samples. Compare expression levels with clinical outcomes data to establish correlations with progression and survival .

• Subcellular localization studies: Employ immunofluorescence co-localization studies with centrosomal markers (e.g., γ-tubulin) to examine potential alterations in MZT2A localization in cancer cells.

• Functional pathway analysis: Use Western blotting with phospho-specific antibodies to investigate MZT2A's reported effect on Akt phosphorylation in cancer cells. Compare signaling in cells with normal versus altered MZT2A expression .

• Experimental workflow for NSCLC studies:

  • Transfect cells with MZT2A-specific siRNA or overexpression constructs

  • Validate knockdown/overexpression by Western blot with MZT2A antibodies

  • Assess effects on proliferation, invasion, and microtubule organization

  • Analyze downstream molecular changes, particularly in the Akt pathway and LGALS3BP expression

• In vivo model evaluation: Use MZT2A antibodies for immunohistochemical analysis of xenograft tumors derived from cells with manipulated MZT2A expression to correlate with tumor growth characteristics .

What methodological approaches enable investigation of MZT2A interactions with the γ-tubulin ring complex?

To study MZT2A's interactions with the γ-tubulin ring complex (γ-TuRC), implement these advanced methodological approaches:

• Co-immunoprecipitation protocol:

  • Lyse cells in non-denaturing buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, with protease inhibitors

  • Pre-clear lysate with protein A/G beads

  • Incubate with anti-MZT2A antibody overnight at 4°C

  • Add protein A/G beads and incubate 2-4 hours

  • Wash beads extensively with lysis buffer

  • Elute and analyze by Western blot using antibodies against γ-TuRC components

• Proximity ligation assay (PLA):

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.2% Triton X-100

  • Block with 5% BSA

  • Incubate with rabbit anti-MZT2A and mouse anti-γ-tubulin antibodies

  • Proceed with PLA protocol using appropriate PLA probes

  • Visualize interaction signals (typically appearing as fluorescent dots) by confocal microscopy

• Domain mapping experiments:

  • Express MZT2A mutants with modifications to the MOZART2 domain

  • Use constructs similar to those described in literature: MZT2A-ΔMOZART2 (with MOZART2 domain knocked out) and MZT2A-MOZART2 (containing primarily the MOZART2 domain)

  • Assess interaction with γ-TuRC components through co-immunoprecipitation

  • Evaluate functional consequences on microtubule nucleation activity

How can researchers design experiments to investigate MZT2A's influence on Akt signaling pathways?

Based on research showing MZT2A's role in Akt pathway regulation, particularly in cancer contexts, researchers can implement this experimental approach:

• Experimental design for MZT2A-Akt pathway investigation:

  A. Genetic manipulation setup:

  • Generate cellular models with:

    • MZT2A overexpression (wild-type)

    • MZT2A domain-specific mutants (MOZART2 domain)

    • MZT2A knockdown (siRNA)

  • Confirm manipulation success via Western blot with anti-MZT2A antibodies

  B. Signaling pathway assessment:

  • Analyze Akt phosphorylation status using phospho-specific antibodies (p-Akt)

  • Assess levels of LGALS3BP (reported downstream effector)

  • Include treatments with Akt inhibitor (e.g., LY294002) as controls

  C. Functional assays:

  • Cell viability assays (MTT or similar)

  • Invasion assays (Transwell or similar)

  • Cell cycle analysis (flow cytometry)

• Design of rescue experiments:

  • In MZT2A-knockdown cells, re-introduce:

    • Wild-type MZT2A

    • MOZART2 domain mutants

  • Assess whether wild-type, but not mutants, rescues Akt phosphorylation

• Correlation analysis in patient samples:

  • Perform immunohistochemistry on patient-derived samples using:

    • Anti-MZT2A antibodies

    • Anti-phospho-Akt antibodies

    • Anti-LGALS3BP antibodies

  • Analyze correlation between MZT2A expression, Akt phosphorylation, and clinical outcomes

What novel methodologies are being developed for enhanced MZT2A detection and analysis?

Emerging methodologies for MZT2A detection and analysis reflect broader trends in antibody technology advancement:

• Multiplexed imaging approaches:

  • Mass cytometry (CyTOF) adaptations using metal-conjugated anti-MZT2A antibodies for simultaneous detection of multiple centrosomal proteins

  • Multiplexed immunofluorescence using tyramide signal amplification (TSA) to detect low-abundance MZT2A alongside other markers

• Super-resolution microscopy applications:

  • STORM or PALM microscopy with fluorophore-conjugated MZT2A antibodies to resolve nanoscale centrosomal organization

  • Expansion microscopy protocols adapted for centrosomal proteins to physically enlarge structures for enhanced visualization

• Live-cell applications:

  • Development of cell-permeable antibody fragments (nanobodies) against MZT2A for live-cell imaging

  • CRISPR-based tagging of endogenous MZT2A for live visualization without antibodies

• Single-cell proteomics integration:

  • Adaptation of MZT2A antibodies for single-cell Western blotting

  • Application in microfluidic antibody capture techniques for single-cell protein analysis

How might computational approaches enhance antibody design for improved MZT2A specificity?

Recent advances in computational antibody engineering offer promising directions for developing next-generation MZT2A-specific antibodies:

• Epitope mapping and antibody design workflow:

  • Computational prediction of MZT2A-specific epitopes that distinguish it from related proteins

  • Structure-based antibody design targeting these unique epitopes

  • In silico affinity maturation to optimize binding properties

• Machine learning applications:

  • Development of models to predict cross-reactivity based on antibody sequence and structural features

  • Training algorithms on experimental binding data to improve specificity predictions

• Biophysics-informed modeling for specificity enhancement:

  • Application of approaches similar to those described in recent literature on antibody specificity inference

  • Identification of different binding modes associated with particular MZT2A conformations

  • Computational design of antibodies with customized specificity profiles for MZT2A

• Integration of experimental and computational validation:

  • Phage display optimization guided by computational predictions

  • High-throughput sequencing and downstream computational analysis to identify optimal binders

  • Validation of designed antibodies against recombinant MZT2A variants

What considerations are important when designing long-term studies tracking MZT2A expression in disease progression?

For longitudinal studies of MZT2A in disease contexts, researchers should implement these methodological considerations:

• Antibody consistency and validation strategy:

  • Reserve sufficient quantities of the same antibody lot for the entire study duration

  • Perform periodic validation to ensure consistent performance over time

  • Include internal controls in each batch of experiments to normalize between time points

• Sample collection and preservation protocol:

  • Standardize fixation methods and times for tissue samples

  • Consider tissue microarrays for simultaneous processing of multiple timepoints

  • Implement rigorous sampling procedures to ensure representative tissue collection

• Quantification methods:

  • Establish objective scoring systems for immunohistochemistry

  • Use digital pathology and automated image analysis for consistent quantification

  • Include multiple observers for subjective assessments to reduce bias

• Data integration framework:

  • Correlate MZT2A expression with other molecular markers, particularly phospho-Akt and LGALS3BP

  • Integrate with clinical data using appropriate statistical methods

  • Consider time-dependent statistical analyses for progression studies

• Consideration of antibody dynamics in longitudinal serum studies:

  • If developing assays for detecting circulating MZT2A, consider lessons from studies on antibody persistence in other conditions

  • Recognize that antibody detection may remain effective for extended periods (>1 year) based on similar studies