mynn Antibody

What is Myoneurin (MYNN) and why is it a target for antibody development?

Myoneurin (MYNN), also known as Zinc finger and BTB domain-containing protein 31 (ZBTB31), is a protein involved in transcription regulation with a molecular mass of approximately 78 kDa . As a member of the zinc finger protein family with BTB domains, MYNN plays important roles in gene expression regulation. The development of antibodies targeting MYNN enables researchers to study its expression patterns, localization, and interactions in various biological contexts.

The protein has several aliases including OSZF, ZBTB31, and SBBIZ1, which may appear in literature and antibody documentation . When designing experiments involving MYNN antibodies, researchers should be aware of these alternative designations to ensure comprehensive literature searches and proper antibody selection.

How should I determine the optimal antibody concentration for MYNN detection in different applications?

The optimal antibody concentration varies by application and antibody format. A methodical titration approach is recommended:

For IHC-P applications:

• Begin with the manufacturer's recommended range (typically 1-20 μg/ml)

• Published data shows successful MYNN detection in human placenta tissue at 1/100 dilution

• Perform a dilution series (e.g., 1:50, 1:100, 1:200, 1:500)

• Include appropriate positive and negative controls

• Evaluate signal-to-noise ratio at each concentration

For Western blotting:

• Start with 2 μg/ml as this concentration has been validated in published protocols

• Perform systematic titration if signal quality is suboptimal

• Consider protein loading amounts (10-30 μg total protein per lane) when optimizing

Remember that antibody affinity can vary between lots and manufacturers, necessitating optimization for each new antibody .

What are the key considerations for sample preparation to ensure optimal MYNN epitope preservation?

MYNN epitope preservation requires careful attention to fixation and extraction procedures:

For tissue sections (IHC-P):

• Standard formalin fixation (10% neutral buffered formalin for 24-48 hours) has been validated

• Antigen retrieval methods may be necessary—heat-induced epitope retrieval in citrate buffer (pH 6.0) is often effective

• Overfixation can mask epitopes; adhere to validated protocols

For protein extracts (WB):

• Use lysis buffers containing protease inhibitors to prevent degradation

• For nuclear proteins like MYNN, consider specialized nuclear extraction protocols

• Flash-freeze tissues immediately after collection to minimize protein degradation

• Store lysates at -80°C and avoid repeated freeze-thaw cycles

The immunogen used to generate most MYNN antibodies corresponds to amino acids 50-350 or recombinant fragments, so preservation of this region is particularly important .

How can I validate the specificity of a MYNN antibody for my experimental system?

Multi-level validation strategies are essential for confirming antibody specificity:

• Primary Validation Methods:

  • Positive and negative tissue controls based on known MYNN expression patterns

  • Peptide competition assays using the immunizing peptide

  • Knockdown/knockout validation using siRNA or CRISPR-Cas9 targeting MYNN

  • Testing in multiple applications (IHC, WB, IF) for consistent detection patterns

• Specialized Validation Approaches:

  • Comparison of staining patterns using multiple antibodies targeting different MYNN epitopes

  • Correlation of protein detection with mRNA expression data

  • Mass spectrometry confirmation of immunoprecipitated proteins

As emphasized in FDA guidance on immunoassay validation, specificity refers to the ability to exclusively detect the target analyte, avoiding false positives that could obscure relationships in experimental data .

What controls should be included when using MYNN antibodies in immunoassays?

A comprehensive control strategy includes:

The FDA guidance on assay development emphasizes that controls should be designed to detect antibodies that could mediate unwanted biological consequences, which is relevant for research applications requiring high specificity .

How can MYNN antibodies be incorporated into multiplex immunofluorescence studies?

Multiplexing with MYNN antibodies requires careful antibody panel design:

• Antibody Selection Criteria:

  • Select antibodies raised in different host species to enable discrimination with species-specific secondary antibodies

  • For rabbit polyclonal anti-MYNN antibodies , pair with mouse, goat, or guinea pig antibodies against other targets

  • Consider direct conjugation of the MYNN antibody (e.g., biotin-conjugated variants are available )

• Epitope Preservation Strategy:

  • Use a sequential staining approach if antigen retrieval requirements differ between targets

  • Test compatibility of multiplex conditions with single-plex controls

  • Consider tyramide signal amplification for weak signals

• Spectral Considerations:

  • Choose fluorophores with minimal spectral overlap

  • Include single-color controls for spectral unmixing if using multispectral imaging

  • Use appropriate autofluorescence quenching techniques

When designing multiplex panels, consider that biotin-conjugated MYNN antibodies offer flexibility for detection with various streptavidin-fluorophore conjugates, enabling strategic positioning within the spectral range.

What approaches can be used to assess potential cross-reactivity with other BTB domain-containing proteins?

Cross-reactivity assessment is crucial for BTB domain proteins due to structural similarities:

• Computational Approaches:

  • Sequence homology analysis comparing the immunogen sequence (aa 50-350) to other BTB domain proteins

  • Epitope prediction software to identify potential shared epitopes

• Experimental Validation:

  • Western blot analysis of recombinant BTB domain proteins with similar molecular weights

  • Testing in tissues with differential expression of BTB family members

  • Competitive binding assays with recombinant BTB domain proteins

• Advanced Techniques:

  • Surface plasmon resonance (SPR) to measure binding affinities to different BTB proteins

  • Mass spectrometry analysis of immunoprecipitated complexes

Research on natural monoclonal antibodies has demonstrated that antibody specificity and affinity do not always correlate, with KDs for macromolecules ranging between 10^-5 and 10^-10 M . This principle applies to polyclonal antibodies as well, necessitating thorough cross-reactivity assessment.

What are the most common causes of false-negative results when detecting MYNN, and how can they be addressed?

False-negative results may stem from several factors:

• Epitope Masking and Accessibility Issues:

  • MYNN is a nuclear protein; ensure nuclear membrane permeabilization in IF/IHC

  • Optimize antigen retrieval methods (heat-induced vs. enzymatic)

  • Try alternative fixation protocols if formalin fixation yields negative results

• Technical Limitations:

  • Drug interference: Sample collection timing can affect results if therapeutic proteins are present

  • Matrix interference: Optimize Minimal Required Dilution (MRD) to minimize matrix effects

  • Antibody concentration: Titrate to identify optimal working concentration

• Biological Variables:

  • Expression levels: MYNN expression may be tissue/condition specific

  • Protein modification: Post-translational modifications may mask epitopes

  • Protein degradation: Ensure appropriate sample handling and storage

When troubleshooting, consider using positive control tissues with known MYNN expression, such as human placenta, which has been validated for MYNN detection .

What strategies can improve signal detection in tissues with low MYNN expression?

For detecting low-abundance MYNN expression:

• Signal Amplification Methods:

  • Tyramide signal amplification (TSA) can increase sensitivity 10-100 fold

  • Polymer-based detection systems improve signal compared to traditional ABC methods

  • Consider biotin-conjugated primary antibodies for streptavidin-based amplification

• Optimization Approaches:

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

  • Reduce washing stringency (shorter washes, milder buffers)

  • Increase antibody concentration within reasonable limits to avoid background

• Technical Enhancements:

  • Use high-sensitivity detection substrates for chromogenic detection

  • Consider fluorescence detection with high-sensitivity cameras

  • Implement computational image analysis to detect subtle signals

Research on antibody development indicates that acid dissociation approaches may improve detection by disrupting antibody-antigen complexes when target proteins are bound to other molecules, potentially applicable to transcription factors like MYNN .

How should I interpret variations in MYNN staining patterns between different cell types or tissues?

Variation in MYNN staining patterns requires careful interpretation:

• Subcellular Localization Analysis:

  • Nuclear localization is expected for MYNN as a transcription factor

  • Cytoplasmic staining should be critically evaluated for specificity

  • Co-localization with other nuclear markers can confirm proper localization

• Expression Level Considerations:

  • Quantify relative expression levels using appropriate image analysis

  • Consider cell cycle-dependent expression patterns

  • Correlate with complementary techniques (qPCR, RNA-seq) for validation

• Context-Specific Interpretation:

  • Different tissues may show variable expression patterns

  • Development and differentiation state may influence expression

  • Disease states may alter expression patterns or localization

When interpreting results, remember that differences in antibody affinity and epitope accessibility can influence apparent expression patterns, necessitating validation with multiple detection methods .

What statistical approaches are appropriate for quantifying MYNN expression in immunohistochemical studies?

Quantification of MYNN expression should employ rigorous statistical methods:

• Semi-quantitative Scoring Systems:

  • H-score (0-300): Intensity (0-3) × percentage of positive cells (0-100%)

  • Allred score: Intensity (0-3) + proportion score (0-5)

  • Nuclear scoring systems specific for transcription factors

• Digital Image Analysis Approaches:

  • Automated cell counting with nuclear recognition algorithms

  • Intensity thresholding with background correction

  • Machine learning-based classification of staining patterns

• Statistical Analysis Guidelines:

  • Non-parametric tests for semi-quantitative data (Mann-Whitney, Kruskal-Wallis)

  • ANOVA for continuous measurements with normal distribution

  • Appropriate multiple testing correction for studies examining multiple tissues

• Reporting Standards:

  • Include both raw data and derived scores

  • Report clear thresholds for positivity

  • Provide representative images of scoring categories

For correlation with clinical or experimental variables, approaches similar to those used in antibody response studies may be applicable, including hierarchical linear regressions to determine associations independent of covariates .

How can MYNN antibodies be adapted for single-cell protein analysis techniques?

Emerging single-cell techniques present new opportunities for MYNN analysis:

• Mass Cytometry (CyTOF) Applications:

  • Metal-conjugated MYNN antibodies enable integration into CyTOF panels

  • Allows simultaneous detection with dozens of other proteins

  • Requires validation of metal conjugation effects on binding properties

• Single-Cell Western Blotting:

  • Miniaturized protein separation and antibody probing

  • Enables correlation of MYNN with other proteins at single-cell level

  • Requires optimization of lysis conditions for nuclear proteins

• Proximity Ligation Assays (PLA):

  • Detects protein-protein interactions involving MYNN

  • Single-molecule sensitivity for low-abundance interactions

  • Requires pairs of antibodies targeting MYNN and interaction partners

The development of recombinant single-chain variable fragment antibodies (scFv) and metal-binding domains (scFv:MBD) offers potential for improving molecular immunolabeling of proteins like MYNN, with demonstrated improvements in labeling fidelity over traditional approaches .

What are the considerations for developing custom antibodies against specific MYNN domains or post-translational modifications?

Development of specialized MYNN antibodies requires strategic planning:

• Epitope Selection Strategy:

  • Target functional domains (zinc finger, BTB domain) for structure-function studies

  • Consider known or predicted post-translational modification sites

  • Evaluate sequence conservation across species for cross-reactivity potential

• Production Considerations:

  • Recombinant fragment immunization (aa 50-350 has proven successful)

  • Phospho-specific antibody development requires careful phosphopeptide design

  • Consider affinity purification against the specific domain/modification

• Validation Requirements:

  • Domain-specific antibodies require knockout/mutation validation

  • Modification-specific antibodies need validation with modifying/demodifying enzymes

  • Cross-reactivity assessment with related protein domains is essential

The FDA guidance on antibody development emphasizes that positive control antibodies generated by immunizing animals should be affinity purified using the therapeutic protein product , a principle that applies to research antibody development as well.