PATL2 Antibody

What is PATL2 and why is it important in reproductive research?

PATL2 is a RNA-binding protein that acts as a translational repressor belonging to the PAT1 protein family. In humans, the canonical protein has a reported length of 543 amino acid residues and a molecular mass of 61.5 kDa . PATL2 is highly expressed in oocytes and plays a crucial role in oocyte maturation . The importance of PATL2 in reproductive research stems from its association with oocyte maturation defects, with several studies identifying mutations in the PATL2 gene that lead to female infertility . These findings have positioned PATL2 as a critical target for reproductive biology research, particularly in understanding the molecular mechanisms underlying oocyte development and maturation.

What are the common applications for PATL2 antibodies in research?

PATL2 antibodies are employed in various research applications, with immunofluorescence being the most common methodology . Other frequently used applications include:

• Western Blot - For protein expression quantification and validation of PATL2 variants

• Immunocytochemistry - For cellular localization studies

• Immunohistochemistry - For tissue-specific expression analysis

• Flow Cytometry - For cell-specific expression profiling

• ELISA - For protein quantification in solution

• Proximity Ligation Assays (PLA) - For protein-protein interaction studies

When selecting a PATL2 antibody for research, it's important to verify the validated applications for your specific experimental needs and confirm species reactivity, as PATL2 orthologs have been reported in various model organisms including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken .

How should PATL2 antibodies be validated for research use?

Proper validation of PATL2 antibodies is essential for generating reliable research data. A comprehensive validation approach should include:

• Western Blot Validation: Confirm the antibody detects a protein of the expected molecular weight (61.5 kDa for human PATL2) . Multiple studies have used GAPDH as an internal control when quantifying PATL2 expression via Western blot .

• Specificity Testing: Test the antibody against wild-type and mutant PATL2 proteins. Research has shown that mutations such as S459Y, P510T, V401F, R402W, and E428Dfs*9 can affect PATL2 protein stability and expression levels .

• Subcellular Localization Confirmation: Verify correct subcellular localization using immunofluorescence. PATL2 is reported to localize in both the nucleus and cytoplasm .

• Positive and Negative Controls: Include appropriate controls such as PATL2-overexpressing cells (positive control) and PATL2-knockdown cells (negative control) .

• Cross-reactivity Assessment: Test for cross-reactivity with related proteins, especially other PAT1 family members, to ensure specificity .

A rigorous validation strategy will ensure that experimental results accurately reflect PATL2 biology rather than artifacts of non-specific antibody binding.

What are the optimal storage and handling conditions for PATL2 antibodies?

To maintain PATL2 antibody integrity and performance over time, researchers should adhere to these storage and handling guidelines:

• Storage Temperature: Most PATL2 antibodies should be stored at -20°C for long-term storage, with aliquots kept at 4°C for active use to minimize freeze-thaw cycles .

• Aliquoting: Divide stock solutions into small, single-use aliquots to prevent protein degradation from repeated freeze-thaw cycles.

• Dilution Buffer: For working solutions, use appropriate buffers as recommended by manufacturers. For Western blot applications, TBS-T (Tris-buffered saline with Tween) is commonly used for PATL2 antibody dilutions .

• Dilution Factors: Optimal dilution factors vary by application and manufacturer but typically range from 1:1000 to 1:10000 for Western blot applications .

• Secondary Antibody Selection: Choose appropriate species-matched secondary antibodies. For PATL2 detection, goat anti-mouse or anti-rabbit IgG HRP-linked secondary antibodies are commonly used at dilutions around 1:10000 .

Proper storage and handling are crucial for maintaining antibody performance and ensuring reproducible experimental results in PATL2 research.

How can PATL2 antibodies be used to investigate protein-protein interactions in oocyte development?

PATL2 interacts with several key proteins involved in cell cycle regulation and oocyte maturation. Advanced methodologies for investigating these interactions include:

• Co-immunoprecipitation (Co-IP) followed by Mass Spectrometry: This approach has successfully identified 1221 proteins that interact with wild-type PATL2 . For Co-IP experiments, HA-tagged PATL2 constructs can be used to pull down interaction partners, which are then identified via mass spectrometry .

• Proximity Ligation Assay (PLA): PLA has been effectively employed to detect and quantify interactions between PATL2 and CDC23 in situ . This method provides spatial information about protein interactions within cells. Studies have shown significant PLA spots between wild-type HA-PATL2 and FLAG-CDC23, with reduced interaction when using PATL2 mutants like V401F/R402W .

• Immunofluorescence Co-localization: Dual-staining with PATL2 antibodies and antibodies against potential interaction partners can provide preliminary evidence of protein-protein interactions. For example, FLAG-CDC23 and HA-PATL2 have been shown to display uniform distribution in both nucleus and cytoplasm of oocytes .

A comprehensive analysis comparing wild-type and mutant PATL2 interactomes revealed that PATL2 mutations can significantly alter protein-protein interactions. For instance, 436 proteins demonstrated decreased interaction intensity with the PATL2 V401F/R402W mutant compared to wild-type PATL2 . Key interaction partners include CDC23, MAD2L1, and APC1, which are involved in critical cell cycle processes such as sister chromatid segregation and meiotic cell cycle regulation .

What experimental approaches can detect changes in PATL2 expression and localization in mutant versus wild-type oocytes?

To accurately assess the impact of PATL2 mutations on protein expression and localization, researchers can employ these sophisticated approaches:

• Quantitative Immunofluorescence: This technique allows for precise measurement of PATL2 protein levels and subcellular distribution. Studies have shown that mutations such as c.1247C>A cause significant decreases in PATL2 expression in the cytoplasm compared to wild-type PATL2 . When performing quantitative immunofluorescence:

  • Use standardized image acquisition parameters

  • Employ automated image analysis software for unbiased quantification

  • Include appropriate controls for background subtraction and normalization

• Lentiviral Overexpression Systems: Transfection of HEK 293T cells with lentiviral overexpression plasmids containing wild-type or mutant PATL2 allows for direct comparison of expression patterns . This approach has demonstrated that mutations can significantly downregulate both mRNA and protein expression of PATL2 .

• Microinjection of Tagged Constructs: Microinjection of HA-tagged PATL2 (wild-type or mutant) cRNAs into mouse GV oocytes enables direct visualization of protein localization and stability in relevant cell types . This approach has revealed that while mutations may not alter subcellular localization, they can significantly decrease protein levels .

• RT-qPCR for Transcript Quantification: Real-time quantitative PCR can measure changes in PATL2 mRNA levels. Studies have demonstrated that some mutations affect mRNA expression levels, while others primarily impact protein stability without altering transcript levels .

How can PATL2 antibodies be utilized in functional rescue experiments to validate mutation pathogenicity?

Functional rescue experiments are crucial for establishing causality between PATL2 mutations and observed phenotypes. A methodical approach includes:

• siRNA-Mediated Knockdown: First, establish a knockdown model using siRNAs targeting endogenous PATL2 (or Patl2 in mouse models). Confirm knockdown efficiency via RT-PCR and immunofluorescence using PATL2 antibodies .

• Phenotype Characterization: Document the phenotypic consequences of PATL2 depletion. Research has shown that si-Patl2 treatment in mouse oocytes leads to decreased MII oocyte rates without affecting GV breakdown .

• Rescue Construct Design: Generate expression constructs for wild-type and mutant PATL2 variants. HA-tagged constructs are commonly used to differentiate exogenous from endogenous protein .

• Rescue Experiment: Microinject cRNAs encoding wild-type or mutant PATL2 into knockdown oocytes and assess their ability to rescue the phenotype. Studies have demonstrated that:

  • Wild-type HA-PATL2 successfully rescues MII rates in si-Patl2 treated oocytes

  • Missense mutants show reduced rescue capacity

  • Frameshift variants (E428Dfs9 and F539Cfs19) completely fail to rescue oocyte maturation

• Protein Expression Verification: Use PATL2 antibodies to confirm expression of rescue constructs via Western blot or immunofluorescence .

This rescue approach provides strong functional evidence for mutation pathogenicity and has been successfully employed to demonstrate that PATL2 mutations result in varying degrees of protein instability and functional loss .

What methodological considerations are important when using PATL2 antibodies to study mRNA regulation in oocytes?

PATL2's role as an RNA-binding protein that regulates mRNA fate requires specialized approaches when studying its function:

• RNA Immunoprecipitation (RIP): To identify mRNAs bound by PATL2, perform RIP using validated PATL2 antibodies followed by RNA-seq. This approach can identify direct mRNA targets of PATL2, particularly those involved in cell cycle regulation .

• RNA-seq of PATL2 Mutant Oocytes: Compare transcriptomes of wild-type and PATL2 mutant oocytes to identify dysregulated mRNAs. Research has revealed that PATL2 mutations affect mRNA accumulation and translation of key cell cycle-related transcripts .

• Polysome Profiling: To assess PATL2's impact on mRNA translation, combine polysome fractionation with RT-qPCR or RNA-seq to identify transcripts with altered translation efficiency in PATL2 mutant versus wild-type oocytes .

• Dual Immunofluorescence for PATL2 and P-body/Stress Granule Markers: As a translational repressor, PATL2 may localize to RNA processing bodies. Co-staining for PATL2 and markers of P-bodies or stress granules can provide insight into the mechanism of translational repression .

• Spatial Transcriptomics: For spatial resolution of PATL2-regulated mRNAs, combine PATL2 immunofluorescence with in situ hybridization for target mRNAs to visualize co-localization patterns within oocytes.

When interpreting results from these experiments, it's important to consider that PATL2 mutations lead to oocyte meiosis defects through multiple mechanisms: affecting mRNA accumulation, altering mRNA translation, and disrupting direct binding to and stabilization of specific transcripts .

How can PATL2 antibodies be optimized for detecting low expression levels in non-oocyte tissues?

While PATL2 is highly expressed in oocytes, detecting its expression in other tissues where it may be present at lower levels requires methodological optimization:

• Signal Amplification Systems: Consider using tyramide signal amplification (TSA) or other enzymatic amplification methods to enhance detection sensitivity while maintaining specificity.

• Antibody Concentration Optimization: Perform titration experiments with increasing antibody concentrations to determine the optimal dilution that maximizes specific signal while minimizing background. Start with manufacturer recommendations and adjust based on empirical results .

• Extended Incubation Protocols: For low-abundance targets, extending primary antibody incubation times (overnight at 4°C) can improve signal detection without significantly increasing background.

• Sample Preparation Optimization: For tissues with potential masking epitopes, evaluate different antigen retrieval methods (heat-induced vs. enzymatic) to maximize epitope accessibility.

• Alternative Detection Systems: For Western blot applications, consider using more sensitive detection substrates such as chemiluminescent substrates with longer emission half-lives or fluorescent secondary antibodies with direct scanning.

• Pre-enrichment Strategies: For extremely low-abundance detection, consider immunoprecipitation of PATL2 prior to Western blot analysis to concentrate the target protein.

• Validation Controls: Include positive controls (oocyte samples) alongside test samples to confirm assay performance and appropriate antibody function .

This optimized approach can help detect PATL2 in tissues where it may have previously unrecognized functions beyond its established role in oocyte development.

What are common technical issues when using PATL2 antibodies and how can they be resolved?

Researchers may encounter several technical challenges when working with PATL2 antibodies. Here are systematic approaches to troubleshoot common issues:

• High Background Signal:

  • Problem: Non-specific binding leading to high background in immunofluorescence or Western blot

  • Solutions:

    • Increase blocking time and concentration (5% BSA or milk in TBS-T)

    • Optimize antibody dilution through titration experiments

    • Include additional washing steps with increased detergent concentration

    • Pre-absorb antibody with cell/tissue lysates from PATL2-negative samples

• Weak or Absent Signal:

  • Problem: Insufficient antibody binding or protein degradation

  • Solutions:

    • Verify protein isolation protocol preserves PATL2 integrity

    • Include protease inhibitors during sample preparation

    • Optimize antigen retrieval methods for fixed tissues

    • Increase antibody concentration or incubation time

    • Consider signal amplification systems

• Multiple Bands on Western Blot:

  • Problem: Potential detection of isoforms, degradation products, or non-specific binding

  • Solutions:

    • Compare band patterns with positive controls expressing known PATL2 variants

    • Verify specificity using PATL2 knockdown samples

    • Optimize SDS-PAGE conditions for better separation

    • Consider using gradient gels for improved resolution

• Inconsistent Immunoprecipitation Results:

  • Problem: Variable pull-down efficiency affecting protein-protein interaction studies

  • Solutions:

    • Optimize antibody-to-bead ratio and incubation conditions

    • Validate IP efficiency using Western blot of input, supernatant, and IP fractions

    • Consider using tagged PATL2 constructs with tag-specific antibodies for more consistent results

• Discrepancies Between Antibody Lots:

  • Problem: Batch-to-batch variability affecting experimental reproducibility

  • Solutions:

    • Validate each new antibody lot against previous lots using positive controls

    • Purchase larger quantities of validated lots for long-term studies

    • Consider monoclonal antibodies for greater consistency

Implementing these troubleshooting strategies can significantly improve the reliability and reproducibility of PATL2 antibody-based experiments.

How should researchers design experiments to study PATL2 mutations and their effects on protein function?

A comprehensive experimental design for investigating PATL2 mutations should include multiple complementary approaches:

• Expression Vector Construction:

  • Generate mammalian expression vectors containing wild-type and mutant PATL2 sequences

  • Include epitope tags (HA, FLAG) to facilitate detection and distinguish from endogenous protein

  • Consider using lentiviral systems for stable expression

• Cellular Models:

  • HEK293T cells are commonly used for initial characterization

  • For physiologically relevant models, consider:

    • Mouse GV oocytes for microinjection experiments

    • Human cell lines with CRISPR/Cas9-engineered mutations

• Multi-level Assessment Strategy:

  • Transcript Analysis: RT-qPCR to measure mRNA expression levels

  • Protein Expression: Western blot with validated PATL2 antibodies

  • Subcellular Localization: Immunofluorescence to determine if mutations alter protein distribution

  • Protein Stability: Cycloheximide chase assays to measure protein half-life

  • Protein-Protein Interactions: Co-IP, PLA, or MS/MS to identify altered interaction networks

  • Functional Assays: Oocyte maturation rescue experiments

• Experimental Controls:

  • Include wild-type PATL2 as positive control

  • Use empty vector as negative control

  • Include previously characterized PATL2 mutations (L189R, Y217N, I318T) as reference points

• Data Analysis Framework:

  • Quantify protein levels relative to GAPDH or other housekeeping proteins

  • Use statistical analyses appropriate for the experimental design (e.g., one-way ANOVA with multiple-comparison test at 95% confidence)

  • For interaction studies, set clear thresholds for significant changes (e.g., at least 30% reduction in binding intensity)

This systematic approach has successfully demonstrated that mutations such as S459Y and P510T lead to PATL2 protein degradation due to reduced stability, resulting in decreased PATL2 expression levels and ultimately oocyte maturation arrest .

How are PATL2 antibodies being used to explore novel roles beyond oocyte maturation?

While PATL2's role in oocyte maturation is well-established, emerging research is uncovering additional functions:

• PATL2 in mRNA Translation Regulation:

  • Research using PATL2 antibodies has revealed that beyond its role as a translational repressor, PATL2 affects the mRNA accumulation and translation of cell cycle-related proteins .

  • Using PATL2 antibodies in RNA immunoprecipitation followed by sequencing (RIP-seq) can identify the broader mRNA targets of PATL2 in various cell types.

• PATL2 in Cell Cycle Regulation:

  • Immunoprecipitation studies have identified interactions between PATL2 and key cell cycle regulators including CDC23, APC1, and MAD2L1 .

  • These interactions suggest PATL2 may have broader roles in cell cycle regulation beyond oocytes.

• PATL2 in RNA Processing Bodies:

  • As a translational repressor, PATL2 may function in RNA processing bodies or stress granules.

  • Co-localization studies using PATL2 antibodies together with markers of these structures can reveal mechanistic insights.

• Evolutionary Conservation of PATL2 Function:

  • PATL2 orthologs have been identified in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken .

  • Comparative studies using species-specific PATL2 antibodies can elucidate evolutionarily conserved and divergent functions.

Future research directions should explore these potential functions using PATL2 antibodies in diverse experimental contexts beyond reproductive biology.

What standards should be applied when selecting PATL2 antibodies for novel applications?

As PATL2 research expands into new applications, rigorous antibody selection criteria become increasingly important:

• Application-Specific Validation:

  • Verify that the antibody has been validated for your specific application (WB, IF, IHC, IP, etc.)

  • For novel applications, conduct preliminary validation experiments comparing multiple antibodies

• Epitope Considerations:

  • For full-length protein detection, select antibodies targeting conserved regions

  • For mutation studies, choose antibodies whose epitopes do not overlap with the mutation site

  • C-terminal region antibodies may be particularly useful for some applications

• Clonality Selection:

  • Monoclonal antibodies offer higher reproducibility and specificity

  • Polyclonal antibodies may provide stronger signals due to multiple epitope recognition

  • Consider using multiple antibodies recognizing different epitopes for validation

• Species Reactivity:

  • Verify cross-reactivity with the species of interest

  • For evolutionary studies, select antibodies targeting highly conserved epitopes

• Reporting Standards:

  • Document complete antibody information in publications:

    • Manufacturer and catalog number

    • Clone identification for monoclonals

    • Host species and immunogen details

    • Validation methods specific to your application

    • Dilution factors and incubation conditions

• Reproducibility Considerations:

  • For long-term studies, purchase antibodies from established manufacturers with consistent production methods

  • Consider creating a laboratory reference standard to normalize between antibody lots

Adherence to these standards will improve data quality and reproducibility in PATL2 research as the field expands.

What are the current limitations of PATL2 antibody research and future development needs?

Despite significant advances in PATL2 research, several limitations remain in current antibody technologies and applications:

• Current Limitations:

  • Limited availability of mutation-specific antibodies that could differentiate between wild-type and mutant PATL2 proteins

  • Insufficient validation for detecting endogenous PATL2 at physiological levels in non-oocyte tissues

  • Incomplete characterization of antibody cross-reactivity with other PAT family members

  • Variability in antibody performance across different experimental conditions

• Future Development Needs:

  • Development of phospho-specific antibodies to study potential post-translational regulation of PATL2

  • Generation of conformation-specific antibodies that could detect structural changes associated with RNA binding

  • Creation of antibodies suitable for super-resolution microscopy to study PATL2 localization at nanoscale resolution

  • Development of antibody pairs optimized for proximity ligation assays to study specific PATL2 protein-protein interactions in situ

• Methodological Advances Needed:

  • Standardized protocols for PATL2 detection in difficult-to-study samples such as mature oocytes and early embryos

  • Improved techniques for quantifying PATL2-associated mRNPs (messenger ribonucleoproteins)

  • Development of PATL2 activity assays that could measure translational repression function

As research on PATL2's role in oocyte maturation and potentially other cellular processes progresses, these technological advancements will be crucial for deeper mechanistic understanding of PATL2 function in both normal physiology and disease states.

How can researchers integrate PATL2 antibody data with other omics approaches?

To develop a comprehensive understanding of PATL2 function, researchers should employ multi-omics integration strategies:

• Integrating Antibody-Based Data with Transcriptomics:

  • Combine PATL2 immunoprecipitation with RNA-seq to identify directly bound transcripts

  • Correlate PATL2 protein levels (detected by antibodies) with transcriptome changes in mutant vs. wild-type oocytes

  • This approach has already revealed that PATL2 mutations affect the mRNA accumulation and translation of cell cycle-related proteins

• Proteomics Integration:

  • Combine PATL2 antibody-based interactome studies with global proteomics

  • Mass spectrometry analysis following PATL2 immunoprecipitation has identified over 1200 potential interaction partners

  • Differential analysis between wild-type and mutant PATL2 interactomes has revealed 436 proteins with decreased interaction in the presence of mutations

• Functional Genomics Correlation:

  • Integrate CRISPR-based functional screens with PATL2 antibody data

  • Correlate genetic dependencies with PATL2 expression or localization patterns

• Structural Biology Connection:

  • Link antibody epitope mapping data with structural predictions and functional domains

  • Correlate mutation effects on protein structure with changes in antibody recognition and protein function

• Clinical Data Integration:

  • Correlate PATL2 expression patterns with clinical outcomes in infertility cases

  • Studies have already linked specific PATL2 mutations (S459Y, P510T, V401F, R402W) with oocyte maturation defects and female infertility