PBL6 Antibody

What is the PAX6 antibody and what molecular characteristics should researchers be aware of?

The PAX6 antibody is a monoclonal mouse IgG1 with kappa light chain that recognizes the N-terminal region (amino acids 1-223) of the PAX6 protein, a transcription factor critical in neurodevelopment and pancreatic islet cell differentiation. The antibody was deposited to the Developmental Studies Hybridoma Bank (DSHB) by A. Kawakami from the Tokyo Institute of Technology in 1997 . When designing experiments, researchers should account for the antigen's predicted molecular weight of 48 kDa, though it may appear as bands at 38, 40, 46, and 48 kDa in western blots due to post-translational modifications and isoforms . The antibody's epitope has been mapped to the N-terminal region, which is important for designing blocking experiments or when using it in combination with other PAX6 antibodies targeting different epitopes.

What species reactivity has been confirmed for the PAX6 antibody and how should cross-species applications be validated?

The PAX6 antibody has demonstrated remarkably broad cross-species reactivity, making it valuable for comparative studies. Confirmed species reactivity includes:

When applying this antibody to a species not listed above, researchers should first perform validation experiments including positive and negative controls. Western blot validation comparing the observed band pattern with the predicted molecular weight for that species can provide initial confirmation. Additionally, immunohistochemistry on tissues known to express PAX6 (such as developing nervous system or eye) with appropriate blocking controls can further validate cross-species application .

What are the recommended applications for PAX6 antibody and their optimal conditions?

The PAX6 antibody has been validated for multiple experimental applications, each requiring specific optimization:

Each application requires protocol optimization based on specific sample types and experimental conditions . Researchers should always include appropriate positive and negative controls to validate results.

How can computational approaches enhance PAX6 antibody application in complex experimental systems?

Computational approaches similar to those used in antibody-antigen binding studies can significantly enhance PAX6 antibody applications. The Protein Energy Landscape Exploration (PELE) methodology, a Monte Carlo-based approach used for predicting antibody binding efficacy, can be adapted to model PAX6 antibody interactions with target epitopes across different species or mutated versions . This approach involves:

• Three-dimensional modeling of the antibody-epitope binding process

• Application of random translations and rotations to the antigen group

• Perturbations of the protein backbone using normal modes

• Side-chain prediction and energy minimization

• Acceptance or rejection under Metropolis criterion based on total system energy

This computational framework allows researchers to predict binding efficacy prior to experimental validation, particularly useful when working with PAX6 proteins from less-studied species or when investigating the effects of mutations on epitope recognition. For optimal results, simulations should run multiple independent trajectories (144 or more) for at least 48 hours to obtain sufficient sampling of conformational space .

What methodological approaches are recommended for studying PAX6 in neuronal differentiation contexts?

When investigating PAX6's role in neuronal differentiation, researchers should implement a multi-method approach that leverages the PAX6 antibody in both loss-of-function and visualization experiments:

• Temporal expression analysis: Track PAX6 expression during differentiation stages using timed immunofluorescence studies with co-staining for stage-specific markers.

• Function-blocking experiments: Apply PAX6 antibody at various concentrations (5-20 μg/ml) directly to cultures to block protein function, comparing outcomes to isotype controls.

• Chromatin immunoprecipitation sequencing (ChIP-seq): Use PAX6 antibody to identify genome-wide binding sites during different stages of neuronal differentiation.

• Combination with genetic approaches: Compare PAX6 antibody staining patterns in wild-type versus genetically modified systems (CRISPR/Cas9 edits or conditional knockouts).

• Live imaging with tagged secondary antibodies: For non-permeabilized applications to track dynamics in living systems.

This antibody has been specifically noted for its effectiveness in studying how PAX6 controls progenitor cell identity and neuronal fate in response to graded Sonic hedgehog (Shh) signaling , making it valuable for developmental neurobiology research.

How can PAX6 antibody be effectively utilized in chromatin immunoprecipitation experiments?

For effective chromatin immunoprecipitation (ChIP) experiments with PAX6 antibody, researchers should follow this optimized methodology:

• Crosslinking optimization: Test multiple formaldehyde concentrations (0.5-2%) and incubation times (5-20 minutes) as PAX6 binding dynamics can vary by cell type.

• Sonication parameters: Aim for chromatin fragments between 200-500bp, typically requiring 10-15 cycles (30s on/30s off) at medium power.

• Antibody amount: Use 5-10μg PAX6 antibody per ChIP reaction with 25-50μg chromatin.

• Pre-clearing strategy: Pre-clear chromatin with protein A/G beads and non-immune mouse IgG for 1-2 hours to reduce background.

• Incubation conditions: Perform antibody incubation overnight at 4°C with gentle rotation.

• Washing stringency: Include at least one high-stringency wash (with 500mM NaCl) to minimize non-specific binding.

• Validation controls: Always run parallel ChIP with:

  • Non-immune mouse IgG1 (negative control)

  • Antibody against a ubiquitous chromatin mark (positive control)

  • Input samples (0.5-5% of starting material)

• Downstream analysis: For ChIP-seq applications, perform quality control on libraries to ensure sufficient complexity before sequencing.

This approach optimizes the specificity of PAX6 binding detection while minimizing false positives that can occur when transcription factor ChIP protocols are not properly optimized .

What controls are essential when using PAX6 antibody in immunohistochemistry and immunofluorescence studies?

Implementing comprehensive controls is critical for obtaining reliable results with PAX6 antibody in immunohistochemistry (IHC) and immunofluorescence (IF) studies:

For quantitative studies, researchers should additionally include analysis of staining intensity across a dilution series of the primary antibody to ensure working within the linear detection range . This comprehensive control strategy significantly increases confidence in the specificity of observed PAX6 staining patterns.

How should researchers approach epitope retrieval optimization for PAX6 antibody in different sample types?

Epitope retrieval optimization is crucial for PAX6 detection across different sample types due to the effects of fixation on the N-terminal epitope region. The following methodological approach is recommended:

• For formalin-fixed paraffin-embedded (FFPE) tissues:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) for 20 minutes at 95-98°C typically yields optimal results

  • Alternative buffers (Tris-EDTA pH 9.0 or EDTA pH 8.0) should be tested if citrate provides suboptimal results

  • Pressure cooker methods (2-3 minutes at high pressure) often provide more consistent retrieval than water bath methods

• For frozen sections:

  • Mild fixation with 4% paraformaldehyde (10-15 minutes) followed by brief (5-minute) retrieval in citrate buffer

  • Excessive retrieval can damage tissue morphology in frozen sections

• For cultured cells:

  • Fixation method significantly impacts epitope accessibility:

    • 4% paraformaldehyde (10 minutes): Often requires mild retrieval

    • Methanol fixation (-20°C, 10 minutes): May preserve epitope without retrieval

    • Acetone fixation (room temperature, 5 minutes): Often optimal for nuclear transcription factors like PAX6

• Systematic optimization:

  • Test a matrix of conditions including:

    • Multiple retrieval buffers (pH 6.0, 8.0, and 9.0)

    • Different retrieval times (5, 10, 20 minutes)

    • Variable retrieval temperatures (85°C, 95°C, 100°C)

  • Evaluate both signal intensity and preservation of morphology

• Species-specific considerations:

  • The epitope accessibility varies across species; retrieval conditions optimized for mouse samples may not be optimal for human or zebrafish samples

This systematic approach ensures optimal epitope accessibility while maintaining sample integrity, crucial for accurate PAX6 detection across different experimental systems .

What strategies can address batch-to-batch variability in PAX6 antibody performance?

Batch-to-batch variability can significantly impact experimental reproducibility. Researchers should implement these methodological approaches to detect and mitigate variability:

• Performance validation for new batches:

  • Run side-by-side western blots with previous and new antibody batches

  • Compare staining patterns in well-characterized positive control tissues

  • Perform titration experiments to determine if optimal dilutions differ between batches

• Reference standard creation:

  • Prepare a large batch of positive control lysate or fixed tissue sections

  • Aliquot and store under optimal conditions (-80°C for lysates)

  • Use these standards to calibrate each new antibody batch

• Normalization strategies:

  • Implement quantitative normalization using signal intensity ratios between target and housekeeping proteins

  • For immunohistochemistry, use digital image analysis with consistent thresholding parameters

• Documentation and pooling:

  • Maintain detailed records of lot numbers and their performance characteristics

  • For critical long-term projects, consider purchasing and pooling multiple lots at project initiation

• Standardized storage conditions:

  • Store antibody aliquots at -20°C or -80°C to prevent freeze-thaw cycles

  • Use non-frost-free freezers to avoid temperature fluctuations

  • Add carrier proteins (BSA, 0.5-1%) for diluted working stocks

  • Include preservatives like ProClin as recommended by DSHB for long-term storage

• Alternative detection methods:

  • When batch variability is unavoidable, confirm key findings with alternative approaches (RT-PCR, in situ hybridization)

  • Consider using a second PAX6 antibody targeting a different epitope

These strategies significantly reduce the impact of batch variability on experimental outcomes, enhancing reproducibility in PAX6-focused research.

How can researchers resolve non-specific binding and background issues with PAX6 antibody?

Non-specific binding and high background are common challenges when working with PAX6 antibody. This systematic troubleshooting approach addresses these issues:

• Blocking optimization:

  • Test multiple blocking agents:

    • 5-10% normal serum from the species of secondary antibody

    • 3-5% BSA in PBS or TBS

    • Commercial blocking solutions with protein mixtures

  • Extend blocking time to 1-2 hours at room temperature or overnight at 4°C

  • Add 0.1-0.3% Triton X-100 to blocking solution for better penetration

• Washing protocol enhancement:

  • Increase washing steps from 3 to 5 times

  • Extend washing duration to 10 minutes per wash

  • Add 0.1% Tween-20 to wash buffers

  • Use TBS instead of PBS if phospho-epitopes are involved

• Antibody dilution and incubation:

  • Increase antibody dilution incrementally (1:100 → 1:200 → 1:500)

  • Reduce incubation temperature from room temperature to 4°C

  • Add 0.1-0.3% carrier proteins to antibody diluent

  • Consider antibody diluents with background-reducing components

• Sample-specific modifications:

  • For tissues with high endogenous biotin: Add avidin/biotin blocking step

  • For tissues with endogenous peroxidase: Add peroxidase quenching step (3% H₂O₂, 10 minutes)

  • For tissues with high autofluorescence: Add Sudan Black B treatment (0.1% in 70% ethanol)

• Antibody pre-adsorption:

  • Pre-incubate diluted antibody with tissue powder from negative control tissues

  • Centrifuge before use to remove bound non-specific antibodies

• Cross-reactivity analysis:

  • Test the antibody on known negative tissues to identify potential cross-reactive proteins

  • Perform western blots to identify non-specific bands that might indicate cross-reactivity

These approaches should be tested systematically, changing one variable at a time to identify the optimal conditions for reducing background while maintaining specific PAX6 detection .

What are effective strategies for troubleshooting weak or absent PAX6 antibody signals?

When faced with weak or absent signals when using PAX6 antibody, implement this structured troubleshooting approach:

• Sample preparation assessment:

  • Verify PAX6 expression in your sample type through literature or RT-PCR

  • Evaluate fixation parameters (over-fixation can mask epitopes)

  • Confirm proper tissue processing (excessive heat during embedding can denature proteins)

• Epitope retrieval enhancement:

  • Increase retrieval time or temperature incrementally

  • Test alternative retrieval buffers with different pH values

  • For difficult samples, try combined approaches (heat + enzymatic digestion)

• Antibody concentration optimization:

  • Decrease dilution factor (use more concentrated antibody)

  • Extend primary antibody incubation to overnight at 4°C

  • For weak signals, consider two-night incubation with fresh antibody on the second day

• Detection system amplification:

  • Switch from conventional two-step detection to multi-step amplification:

    • Biotin-streptavidin systems

    • Tyramide signal amplification (increases sensitivity 10-100×)

    • Polymerized enzyme detection systems

• Antibody penetration improvement:

  • Increase detergent concentration in buffers (0.1% → 0.3% Triton X-100)

  • For thick sections, extend incubation times or consider vibratome sections

  • For whole-mount samples, implement tissue clearing techniques

• Antibody functionality verification:

  • Test antibody on known positive control (embryonic neural tissue expresses high PAX6 levels)

  • Confirm antibody hasn't degraded by running a western blot with positive control lysate

  • Consider obtaining a fresh antibody aliquot if degradation is suspected

• Species-specific considerations:

  • For non-mammalian models, epitope conservation may be lower; try higher antibody concentrations

  • Consult literature for species-specific optimizations

This methodical approach systematically addresses the most common causes of weak PAX6 staining, leading to significant signal improvement in challenging samples .

What approaches help resolve contradictory results when comparing PAX6 antibody data across different applications?

When faced with contradictory results across different applications using PAX6 antibody, implement this analytical and experimental resolution strategy:

• Epitope accessibility analysis:

  • The N-terminal epitope (aa 1-223) recognized by this antibody may be differentially accessible in various applications

  • In fixed tissues, conformation changes may hide the epitope

  • In denatured western blot samples, the linear epitope becomes fully exposed

  • Create a comparative analysis table documenting epitope accessibility across methods

• Cross-validation with multiple detection methods:

  • Implement at least three independent detection techniques:

    • Protein detection: Western blot, immunoprecipitation, ELISA

    • Localization: Immunohistochemistry, immunofluorescence

    • Expression validation: qRT-PCR, RNA-seq, in situ hybridization

  • Compare results systematically to identify method-specific discrepancies

• Isoform consideration:

  • PAX6 exists in multiple isoforms (including the PAX6 and PAX6(5a) variants)

  • Different methods may preferentially detect specific isoforms

  • Design isoform-specific primers for RT-PCR to correlate with antibody findings

  • Run gradient gels for western blots to better resolve closely migrating isoforms

• Post-translational modification analysis:

  • Phosphorylation or other modifications may affect epitope recognition

  • Compare results from samples treated with phosphatases or other modification-removing enzymes

  • Use modification-specific antibodies alongside the PAX6 antibody to correlate findings

• Experimental variables documentation matrix:

  • Create a comprehensive table documenting all variables across experiments:

    • Sample preparation methods

    • Buffer compositions

    • Incubation conditions

    • Detection systems

  • Systematically harmonize these variables where possible to eliminate technical sources of variation

• Computational binding prediction:

  • Utilize computational approaches similar to those described for antibody-epitope modeling

  • Model the PAX6 epitope under different conditions (native vs. denatured)

  • Predict binding efficacy across experimental conditions

This systematic approach not only resolves contradictions but often reveals biologically meaningful insights about PAX6 structure, modifications, or interactions that explain the observed differences .

How can database resources like PLAbDab enhance PAX6 antibody research and validation?

The Patent and Literature Antibody Database (PLAbDab) and similar resources offer powerful capabilities for advancing PAX6 antibody research through these methodological approaches:

• Comparative sequence analysis:

  • PLAbDab contains over 150,000 paired antibody sequences from more than 10,000 studies

  • Researchers can compare PAX6 antibody sequences with other anti-transcription factor antibodies

  • Identify conserved framework regions and variable CDR patterns that correlate with high specificity

  • Generate multiple sequence alignments to identify structural features contributing to performance

• Structural modeling and optimization:

  • PLAbDab provides 3D structural models for antibodies in its database

  • Compare PAX6 antibody structure with high-performing antibodies targeting similar nuclear proteins

  • Identify structural features that could be modified to enhance performance

  • Use this information to guide humanization or affinity maturation projects

• Literature-based validation approaches:

  • PLAbDab links each antibody to its source documentation

  • Systematically review validation approaches used for similar antibodies

  • Implement best-practice validation protocols used successfully with other nuclear transcription factor antibodies

  • Create a validation matrix based on published approaches

• Alternative antibody identification:

  • Search PLAbDab for alternative PAX6-targeting antibodies or antibodies against related PAX family members

  • Evaluate complementary antibodies targeting different PAX6 epitopes

  • Implement multi-antibody validation strategies based on database findings

• Cross-reactivity prediction:

  • Using PLAbDab's keyword search functionality :

    • Identify potential cross-reactive proteins by sequence similarity

    • Test predicted cross-reactive proteins explicitly in validation experiments

    • Design blocking studies to address predicted cross-reactivity

• Machine learning applications:

  • Apply machine learning algorithms to PLAbDab's extensive antibody dataset

  • Predict antibody performance characteristics based on sequence features

  • Identify optimal conditions for specific applications based on antibody sequence similarities

This strategic use of antibody databases transforms validation from a trial-and-error process to a data-driven approach, significantly enhancing research efficiency and reliability .

How might computational prediction methods improve experimental design with PAX6 antibody?

Computational prediction methods similar to those used for antibody neutralization efficacy can revolutionize experimental design with PAX6 antibody:

• Epitope accessibility prediction:

  • Apply Monte Carlo-based simulation approaches like PELE to predict epitope accessibility

  • Model how different fixation and retrieval methods affect the N-terminal PAX6 epitope

  • Generate heat maps showing predicted binding efficiency across experimental conditions

  • Develop condition-specific optimization matrices based on computational predictions

• Cross-species application prediction:

  • Simulate binding interactions between the PAX6 antibody and target protein from different species

  • Predict relative binding efficiency across species based on epitope conservation

  • Generate species-specific dilution recommendations based on predicted binding energetics

  • Identify species where additional validation is critical due to predicted weak interactions

• Modified protocol development:

  • Simulate how buffer composition affects antibody-epitope interactions

  • Model the effects of detergents, salt concentration, and pH on binding energy landscapes

  • Predict optimal incubation temperature and duration based on association/dissociation kinetics

  • Design application-specific protocols based on these predictions

• Function-blocking experiment design:

  • Model the interaction between PAX6 antibody and its target in the context of DNA binding

  • Predict antibody concentrations needed to achieve functional blocking

  • Identify potential off-target effects through molecular docking with related proteins

  • Design appropriate controls based on predicted binding mechanics

• Integration with experimental validation:

  • Implement an iterative approach combining computational prediction and experimental validation:

    1. Generate initial predictions

    2. Test key predictions experimentally

    3. Refine computational models based on experimental results

    4. Generate new predictions with improved models

This computational-experimental integrated approach can significantly reduce the time and resources needed for protocol optimization while providing deeper mechanistic understanding of antibody-antigen interactions .