MEIKIN Antibody, Biotin conjugated

What is MEIKIN and why is it a significant research target?

MEIKIN (Meiosis-specific kinetochore protein) functions as a key regulator of kinetochore function during meiosis I. It serves dual critical roles: facilitating the mono-orientation of kinetochores on sister chromosomes and protecting centromeric cohesin from separase-mediated cleavage. MEIKIN acts in collaboration with PLK1 and is specifically required for PLK1 enrichment at kinetochores. Importantly, MEIKIN is not required during meiosis II or mitosis, making it a unique target for meiosis-specific studies . Its specialized function in reproductive cell division makes MEIKIN antibodies valuable tools for investigating fertility mechanisms, meiotic errors, and developmental biology.

What are the technical specifications of commercially available MEIKIN Antibody, Biotin conjugated?

The commercially available MEIKIN Antibody, Biotin conjugated is a polyclonal antibody developed using recombinant Human Meiosis-specific kinetochore protein (amino acids 18-91) as the immunogen. The detailed specifications are as follows:

These specifications highlight the antibody's suitability for specific research applications, particularly those requiring biotin-streptavidin detection systems .

How does the biotin conjugation enhance MEIKIN antibody applications compared to unconjugated alternatives?

Biotin conjugation provides several methodological advantages in MEIKIN research applications. The biotin-streptavidin system offers one of the strongest non-covalent interactions in biological systems, being resistant to pH extremes, high salt concentrations, temperature variations, and denaturants .

For MEIKIN detection, this translates to:

• Enhanced sensitivity - The biotin-streptavidin amplification system allows for detection of low-abundance MEIKIN proteins in complex samples

• Versatility in detection systems - Compatibility with multiple secondary detection reagents (streptavidin-HRP, streptavidin-fluorophores)

• Multi-parameter analysis capability - Enables combination with other non-biotin labeled antibodies in multiplex assays

• Reduced background - Less cross-reactivity compared to systems using secondary antibodies

• Stability in experimental conditions - Maintains binding integrity under various buffer conditions used in meiosis research

These advantages must be weighed against potential limitations, including biotin's endogenous presence in some biological samples, which may contribute to background signals in certain applications.

What validation strategies should be employed before using MEIKIN Antibody, Biotin conjugated in critical experiments?

Rigorous validation is essential when working with MEIKIN antibodies, particularly for novel applications beyond manufacturer-verified uses. A comprehensive validation protocol should include:

• Knockout validation: Using MEIKIN knockout cells (such as HAP1 MDK KO) compared to wild-type controls to confirm antibody specificity

• Immunoprecipitation followed by mass spectrometry: To confirm the antibody captures the intended target with minimal off-target binding

• Cross-reactivity testing: Especially important when working with non-human models given the antibody's human reactivity specification

• Application-specific titration: Determining optimal antibody concentration for each application (ELISA, Western blot, immunofluorescence)

• Epitope mapping confirmation: Verifying the antibody recognizes the expected region (aa 18-91) through peptide competition assays

• Reproducibility assessment: Testing across multiple lots if conducting longitudinal studies

This validation framework ensures experimental reliability and facilitates proper interpretation of results in MEIKIN research contexts.

How can MEIKIN Antibody, Biotin conjugated be optimized for immunoprecipitation of MEIKIN-interacting proteins?

Optimizing immunoprecipitation (IP) with MEIKIN Antibody, Biotin conjugated requires careful consideration of several technical factors:

• Bead selection: Use streptavidin-coated magnetic beads rather than agarose for reduced background and higher recovery efficiency

• Pre-clearing strategy: Pre-clear lysates with unconjugated beads to reduce non-specific binding

• Buffer optimization:

  • For nuclear proteins and chromatin-associated complexes: Include 300-400mM NaCl in wash buffers

  • For maintaining MEIKIN-PLK1 interactions: Add phosphatase inhibitors (10mM NaF, 1mM Na₃VO₄)

  • For reducing background: Include 0.1-0.2% Triton X-100 in wash buffers

• Cross-linking consideration: For transient interactions, consider mild formaldehyde cross-linking (0.1-0.3%) before cell lysis

• Elution strategy: Use biotin competition (2-5mM biotin in elution buffer) for native elution or SDS for complete elution

• Controls: Include parallel IPs with:

  • Non-biotinylated control IgG antibodies

  • Samples from MEIKIN-depleted cells

  • Pre-blocking with recombinant MEIKIN protein

When analyzing MEIKIN interactome during meiosis I specifically, synchronization of cell populations is essential for capturing stage-specific interactions .

What are the optimal conditions for using MEIKIN Antibody, Biotin conjugated in flow cytometry applications?

While the manufacturer specifications primarily list ELISA as a verified application , adapting MEIKIN Antibody, Biotin conjugated for flow cytometry requires specific protocol modifications:

• Cell preparation considerations:

  • Fixation: 2-4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilization: 0.1% saponin or 0.3% Triton X-100 for intracellular detection

  • Blocking: 5% BSA with 10% normal serum from non-rabbit species

• Staining parameters:

  • Antibody dilution: Begin with 1:100-1:500 range (requires titration)

  • Incubation: 45-60 minutes at 4°C with gentle agitation

  • Detection: Streptavidin-fluorophore (AF488, PE, or APC depending on panel design)

• Critical controls:

  • Staining of MEIKIN knockout cells as negative control

  • FMO (Fluorescence Minus One) controls for accurate gating

  • Titration series to determine optimal signal-to-noise ratio

• Technical adjustments:

  • Include RNase treatment if co-staining with DNA dyes for cell cycle analysis

  • Extended washing steps (minimum 3 washes) to reduce background from unbound biotinylated antibody

  • Live/dead discrimination dye to exclude non-specific binding to dead cells

For cell cycle-specific analysis of MEIKIN, correlation with DNA content staining provides valuable context given MEIKIN's phase-specific role in meiosis I .

What are common issues when using MEIKIN Antibody, Biotin conjugated in Western blot applications and how can they be resolved?

Researchers often encounter specific challenges when using biotinylated antibodies for MEIKIN detection in Western blots:

When interpreting results, compare findings with knockout controls to validate specific binding patterns . MEIKIN appears at approximately 40kDa, with potential post-translational modifications resulting in mobility shifts.

How should contradictory results between MEIKIN Antibody, Biotin conjugated and other MEIKIN antibodies be interpreted?

When facing discrepancies between results obtained with biotinylated versus non-biotinylated MEIKIN antibodies, systematic investigation should follow this methodological framework:

• Epitope accessibility analysis:

  • The biotin conjugation may mask or alter epitope accessibility

  • Compare epitope regions targeted by different antibodies (e.g., aa 18-91 for the biotinylated version )

  • Consider whether cellular compartmentalization affects accessibility differently

• Binding kinetics evaluation:

  • Biotin conjugation can alter binding affinity or kinetics

  • Perform time-course experiments to assess whether differences are temporal rather than absolute

  • Consider surface plasmon resonance analysis to quantify affinity changes

• Post-translational modification sensitivity:

  • Different antibodies may preferentially recognize distinct phosphorylation states

  • Validate with phosphatase treatment or phospho-specific antibodies

  • Consider whether biotin conjugation affects recognition of modified forms

• Cross-reactivity assessment:

  • Compare specificity using immunoprecipitation followed by mass spectrometry

  • Perform peptide competition assays with recombinant MEIKIN fragments

  • Test in MEIKIN knockout systems to assess non-specific binding profiles

• Methodological validation:

  • Determine if discrepancies are application-specific (WB vs. IP vs. IF)

  • Test different blocking agents to rule out interference with biotin-streptavidin interaction

  • Evaluate buffer compatibility with both antibody types

Integrating data from multiple antibodies targeting different MEIKIN epitopes provides the most comprehensive understanding of this protein's biology and function.

How can MEIKIN Antibody, Biotin conjugated be employed in super-resolution microscopy to study kinetochore architecture during meiosis?

Super-resolution microscopy offers unprecedented insights into MEIKIN localization and kinetochore organization during meiosis. Optimal implementation of MEIKIN Antibody, Biotin conjugated in these advanced applications involves:

• Sample preparation refinements:

  • Fixation: 2% PFA with 0.05% glutaraldehyde preserves nanoscale structures

  • Permeabilization: Gradient permeabilization (0.01-0.1% Triton X-100) maintains delicate nuclear structures

  • Blocking: 3% BSA with 10% normal serum, supplemented with 0.1% saponin

• Technical adaptations for super-resolution techniques:

  • For STORM: Use streptavidin-Alexa Fluor 647 (photoswitchable dye)

  • For STED: Prefer streptavidin-ATTO 647N or STAR 635P

  • For SIM: Any high-quantum yield streptavidin-fluorophore conjugate

  • For DNA-PAINT: Streptavidin-conjugated DNA docking strands

• Co-localization strategy:

  • Primary combination: MEIKIN (biotin) with PLK1 antibody to study functional interaction

  • Secondary markers: CREST antisera to mark centromeres, SYCP3 for synaptonemal complex

  • Use spectral unmixing for multi-protein localization studies

• Analytical considerations:

  • Quantify nearest-neighbor distances between MEIKIN and centromeric markers

  • Measure signal intensity distributions to map concentration gradients

  • Implement cluster analysis to identify MEIKIN-enriched domains

• Biological timing:

  • Use stage-specific markers to analyze MEIKIN recruitment/displacement kinetics

  • Compare localization patterns between meiosis I and II to confirm specificity

This approach enables visualization of MEIKIN's spatial relationship to kinetochore components with 10-20nm resolution, providing mechanistic insights into its role in chromosome segregation during meiosis.

What strategies can be employed to develop a high-throughput screening assay using MEIKIN Antibody, Biotin conjugated to identify compounds affecting meiotic division?

Developing a robust high-throughput screening (HTS) assay using MEIKIN Antibody, Biotin conjugated requires adaptation of traditional techniques to accommodate greater scale and automation:

• Assay platform selection:

  • AlphaScreen technology: Utilizing streptavidin-donor beads with MEIKIN binding partner antibody-acceptor beads

  • High-content imaging: Measuring MEIKIN localization changes in fixed cell arrays

  • ELISA-based quantification: For measuring MEIKIN-interactor binding disruption

• Readout optimization:

  • Primary metric: MEIKIN-PLK1 interaction strength (proximity-based signal)

  • Secondary metrics: MEIKIN localization changes, PLK1 recruitment efficiency

  • Counter-screen: Centromeric cohesin protection assay

• Assay development parameters:

  • Z'-factor optimization: Aim for Z' > 0.5 for robust screening

  • Signal window: Establish positive controls giving >3-fold signal over background

  • Miniaturization: Validate in 384-well format with 20-50μL volumes

  • DMSO tolerance: Establish acceptable DMSO concentration (<1% preferred)

• Validation framework:

  • Orthogonal assays: Confirm hits with non-biotin detection methods

  • Dose-response assessment: 8-point curves for promising compounds

  • Selectivity panel: Test activity against related kinetochore proteins

  • Cellular phenotype confirmation: Analyze chromosome segregation patterns

• Data analysis approach:

  • Machine learning classification of hit compounds

  • Structure-activity relationship building from hit clusters

  • Pathway enrichment analysis of validated hits

This methodology enables screening of 10,000-100,000 compounds to identify modulators of MEIKIN function, potentially revealing novel chemical probes for meiosis research and fertility-related therapeutics development.

How does MEIKIN Antibody, Biotin conjugated compare with other techniques for studying kinetochore function during meiosis?

When evaluating methodological approaches for kinetochore research, MEIKIN Antibody, Biotin conjugated offers specific advantages and limitations relative to alternative techniques:

The optimal approach depends on specific research questions, with MEIKIN Antibody, Biotin conjugated particularly valuable for detecting endogenous protein in fixed samples and for biochemical isolation of MEIKIN-containing complexes .

What are the critical considerations when selecting between different commercial MEIKIN antibodies for specific research applications?

Selection of optimal MEIKIN antibodies should follow a systematic evaluation framework considering multiple technical and experimental factors:

• Epitope-specific considerations:

  • N-terminal targeting antibodies (aa 1-100): Better for detecting full-length protein

  • C-terminal targeting antibodies: Useful for distinguishing potential isoforms

  • Middle region antibodies: May be affected by post-translational modifications

  • Biotin-conjugated antibody epitope (aa 18-91) : Provides versatile detection options

• Application-specific performance metrics:

  • Western blot: Prioritize antibodies validated with knockout controls

  • Immunofluorescence: Select antibodies with demonstrated specificity in fixed tissues

  • ChIP applications: Consider antibodies validated for chromatin immunoprecipitation

  • Flow cytometry: Choose antibodies that maintain reactivity in suspension conditions

• Technical specifications comparison:

  • Clonality: Polyclonal offers broader epitope recognition; monoclonal provides consistency

  • Host species: Consider compatibility with other antibodies in multi-labeling experiments

  • Conjugation: Biotin provides detection flexibility; direct fluorophore conjugation simplifies workflows

  • Validation breadth: Prioritize antibodies tested across multiple methods and cell types

• Experimental design alignment:

  • If studying MEIKIN-PLK1 interaction: Choose antibodies with validated epitopes outside the interaction domain

  • For co-localization studies: Select antibodies raised in different host species than co-staining antibodies

  • For quantitative applications: Select antibodies with demonstrated linear signal response

The comprehensive data for candidate antibody selection should incorporate validation results from systematic studies similar to those conducted for other targets , including direct comparison of staining patterns between wild-type and knockout cells.

How might MEIKIN Antibody, Biotin conjugated facilitate investigation of species-specific differences in meiotic regulation?

MEIKIN Antibody, Biotin conjugated offers methodological advantages for comparative studies across species, despite its primary validation in human systems:

• Cross-species reactivity assessment methodology:

  • Sequence homology analysis: Aligning immunogen region (aa 18-91) across species

  • Western blot validation: Testing antibody across tissue samples from multiple organisms

  • Epitope conservation mapping: Using peptide arrays to identify conserved binding regions

• Evolutionary conservation investigation approach:

  • Immunoprecipitation from multiple species followed by mass spectrometry

  • Comparative immunofluorescence in meiotic cells from different organisms

  • MEIKIN interactome comparison across evolutionary distance

• Methodological adaptations for cross-species work:

  • Buffer optimization for species-specific cellular compositions

  • Fixation protocol modifications for tissue-specific requirements

  • Antigen retrieval customization for species variations in protein folding

• Technical considerations for novel species applications:

  • Validation priorities: Begin with Western blot before moving to more complex applications

  • Sensitivity enhancement: Consider tyramide signal amplification for low homology species

  • Background reduction: Species-specific blocking reagents to minimize non-specific binding

This comparative approach could reveal fundamental conservation or divergence in meiotic regulation mechanisms across evolutionary lineages, potentially identifying species-specific adaptations in chromosome segregation control.

What emerging technologies could be combined with MEIKIN Antibody, Biotin conjugated to advance understanding of meiotic chromosome dynamics?

Integration of MEIKIN Antibody, Biotin conjugated with cutting-edge technologies offers promising avenues for deeper insights into meiotic processes:

• Spatial multi-omics applications:

  • Spatial transcriptomics with MEIKIN protein mapping

  • Methodology: Combined immunofluorescence and in situ sequencing

  • Research potential: Correlating MEIKIN localization with local transcriptional states

• Advanced imaging technologies:

  • Lattice light-sheet microscopy with two-photon activation

  • Technical approach: Labeling with caged fluorophore-conjugated streptavidin

  • Advantage: Reduced phototoxicity for extended live imaging of meiotic progression

• Protein-protein interaction mapping:

  • BiFC (Bimolecular Fluorescence Complementation) combined with proximity ligation

  • Implementation: Secondary antibodies with split fluorescent protein fragments

  • Benefit: Visualization of specific interaction events in their native context

• Single-cell proteomics integration:

  • Antibody-based single-cell protein analysis (CITE-seq adaptation)

  • Method: MEIKIN antibody conjugated to DNA barcodes for single-cell readout

  • Application: Correlating MEIKIN levels with cellular phenotypes across meiotic stages

• Optogenetic manipulation platforms:

  • Targeted protein degradation of MEIKIN interactors

  • Approach: Light-inducible degron systems with MEIKIN localization monitoring

  • Potential: Dissecting temporal requirements for MEIKIN-partner interactions

These technological integrations could transform understanding of MEIKIN's dynamic function in three-dimensional chromosome organization during meiosis, potentially revealing new mechanisms of chromosome segregation regulation with implications for reproductive biology and fertility research.