pals1a Antibody

What is PALS1/MPP5 and why is it significant in research?

PALS1/MPP5 is a member of the peripheral membrane-associated guanylate kinase (MAGUK) family that functions in tumor suppression and receptor clustering . It plays critical roles in tight junction biogenesis and the establishment of cell polarity in epithelial cells. The protein is essential for adherens junction formation by ensuring correct localization of the exocyst complex protein EXOC4/SEC8, which facilitates trafficking of adherens junction structural components like CDH1 to the cell surface . PALS1 is also integral to vascular lumen formation, endothelial membrane polarity, and various developmental processes including retinal development and cerebellar histogenesis . Its involvement in multiple cellular processes makes it a significant target for research across neurobiology, developmental biology, and cancer research.

What are the recommended applications for PALS1/MPP5 antibodies?

Based on validated research protocols, PALS1/MPP5 antibodies can be reliably used in several applications:

• Flow Cytometry (10 μg/mL recommended concentration)

• Immunocytochemistry/Immunofluorescence (10 μg/mL recommended concentration)

• Immunohistochemistry-Paraffin (5 μg/mL recommended concentration)

• Peptide ELISA (detection limit 1:32000)

• Western Blotting (preliminary testing shows a band at approximately 75kDa in Human Cerebellum lysate)

• Immunoprecipitation

Each application requires specific optimization for best results, particularly regarding fixation methods, antigen retrieval, and antibody concentration.

How should PALS1/MPP5 antibodies be stored and handled to maintain efficacy?

For optimal performance, PALS1/MPP5 antibodies should be stored at -20°C and freeze-thaw cycles should be avoided to preserve antibody integrity . Commercially available antibodies are typically formulated in Tris saline (20 mM Tris pH 7.3, 150 mM NaCl) with 0.5% BSA and 0.02% Sodium Azide as a preservative . The recommended concentration is typically 0.5 mg/ml, though this may vary by manufacturer. When shipping is required, the product should be transported with polar packs and immediately stored at the recommended temperature upon receipt .

How should I design experiments to validate antibody specificity for PALS1/MPP5?

Designing experiments to validate PALS1/MPP5 antibody specificity requires a multi-faceted approach:

• Positive and Negative Controls: Include tissues or cell lines known to express PALS1/MPP5 (e.g., HeLa cells, human kidney tissue) as positive controls . For negative controls, use unimmunized IgG followed by the same secondary antibody .

• Cross-Reactivity Testing: Test against closely related proteins within the MAGUK family to ensure specificity.

• Knockout/Knockdown Validation: Use CRISPR-Cas9 knockout or siRNA knockdown of PALS1/MPP5 to confirm specificity.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide to block specific binding sites before application.

• Multiple Detection Methods: Validate findings using different techniques (e.g., IF, WB, IHC) to confirm consistent results across platforms .

A biophysics-informed model can be employed to identify distinct binding modes associated with specific targets, which may help distinguish between close homologs of the protein .

What are the optimal parameters for immunofluorescence staining with PALS1/MPP5 antibodies?

Optimal immunofluorescence staining with PALS1/MPP5 antibodies involves:

• Fixation: Paraformaldehyde fixation (typically 4%) is recommended based on successful protocols .

• Permeabilization: 0.15% Triton X-100 has been effectively used for permeabilizing fixed HeLa cells .

• Antibody Concentration: Primary incubation with 10 μg/mL of antibody for 1 hour at room temperature .

• Secondary Antibody: Alexa Fluor 488-conjugated secondary antibody at 2 μg/mL concentration .

• Nuclear Counterstain: DAPI is commonly used for nuclear visualization .

• Expected Localization: Look for Golgi and cytoplasmic staining patterns, as these are characteristic of PALS1/MPP5 .

• Negative Controls: Always include unimmunized IgG controls at the same concentration as the primary antibody .

Successful immunofluorescence will show distinct subcellular localization patterns, particularly at tight junctions and the Golgi apparatus.

How do I optimize Western blot protocols for PALS1/MPP5 detection?

Optimizing Western blot protocols for PALS1/MPP5 requires attention to several parameters:

• Sample Preparation: Use appropriate lysis buffers that preserve protein structure while effectively extracting membrane-associated proteins.

• Expected Molecular Weight: Look for bands at approximately 75 kDa (calculated MW of 73.4 kDa according to NP_001243479.1) .

• Antibody Concentration: Start with 1 μg/ml concentration for primary antibody incubation .

• Incubation Time and Temperature: Primary incubation for 1 hour at room temperature has shown good results .

• Blocking Conditions: Use 5% non-fat dry milk or BSA in TBST to reduce background.

• Tissue Selection: Human cerebellum lysate has been validated as a suitable positive control .

• Detection System: Choose a detection system appropriate for your research needs (chemiluminescence, fluorescence, etc.).

If multiple bands appear, additional validation may be required to confirm specificity, such as pre-absorption with the immunizing peptide.

How can computational approaches enhance antibody selection for PALS1/MPP5 research?

Computational approaches can significantly enhance antibody selection for PALS1/MPP5 research through several strategies:

• Biophysics-Informed Modeling: This approach associates distinct binding modes with specific ligands, enabling prediction and generation of antibody variants beyond those observed in experiments . Such models can be trained on experimentally selected antibodies to predict outcomes for new ligand combinations .

• Custom Specificity Profiles: Computational design can create antibodies with:

  • High specificity for a particular target ligand

  • Cross-specificity for multiple target ligands

• Energy Function Optimization: Customized binding profiles can be created by optimizing energy functions:

  • For cross-specific sequences: Jointly minimize the energy functions associated with desired ligands

  • For specific sequences: Minimize energy for desired ligands while maximizing it for undesired ligands

• Statistical Approaches for Selection: When dealing with multiple antibody targets, several statistical methods can guide selection:

  • Shapiro-Wilk test to evaluate normality of distribution

  • Finite mixture models for analyzing latent serological populations

  • Wilks's likelihood ratio test to compare models

  • Benjamini-Yekutieli procedure for adjusting p-values in multiple testing scenarios

• Super-Learner Approach: This combines multiple statistical or machine learning models to predict antibody performance, potentially improving AUC scores to 0.801 (95% CI=0.709-0.892) in some studies .

These computational methods can help overcome the challenges of brute-force approaches, which become computationally infeasible when dealing with more than 5 antibody targets .

How does PALS1/MPP5 interaction with pathogens influence experimental design?

PALS1/MPP5 interactions with pathogens, particularly viruses, should inform experimental design in several ways:

• SARS-CoV Protein E Interaction: Research has shown that the carboxy-terminal domain of SARS-CoV E protein binds to human PALS1 . The last four carboxy-terminal amino acids of E form a novel PDZ-binding motif that binds to the PALS1 PDZ domain .

• Subcellular Redistribution: In SARS-CoV-infected cells, PALS1 redistributes to the ERGIC/Golgi region where viral protein E accumulates . This should be considered when analyzing PALS1 localization in infected cells.

• Epithelial Barrier Disruption: SARS-CoV E protein may act as a virulence factor by hijacking PALS1, causing damage to the epithelial barrier . Experimental designs should account for this when studying epithelial integrity.

• Coimmunoprecipitation and Pull-Down Assays: These techniques have successfully demonstrated E-PALS1 interactions in mammalian cells and should be considered for studying similar pathogen-host protein interactions .

• 3D Culture Models: MDCKII epithelial cells expressing SARS-CoV E protein show altered cyst morphogenesis and tight junction formation, suggesting that 3D culture systems may be valuable for studying PALS1 disruption in disease states .

When designing experiments involving pathogens, researchers should consider dual immunolabeling to track both pathogen proteins and PALS1 redistribution, as well as functional assays to measure epithelial barrier integrity.

What methodological considerations are important when studying PALS1/MPP5 in different tissue contexts?

Studying PALS1/MPP5 across different tissue contexts requires tailored methodological approaches:

• Epithelial Tissues: Focus on tight junction and adherens junction formation, where PALS1 plays critical roles in polarity establishment. Techniques should include:

  • Trans-epithelial electrical resistance (TEER) measurements

  • Calcium switch assays to study junction assembly dynamics

  • Co-localization studies with other polarity complex proteins (CRUMBS3, PATJ)

• Neural Tissues: Given PALS1's role in neuronal progenitor cell survival and cerebellar development, consider:

  • BrdU incorporation assays to study proliferation

  • Lineage tracing experiments

  • Analysis of mTOR signaling pathways

• Vascular Endothelium: For studying PALS1's role in vascular lumen formation and endothelial membrane polarity:

  • Co-immunoprecipitation with CDH5

  • Tube formation assays

  • In vivo vascular development models

• Retinal Tissue: For embryonic and postnatal retinal development studies:

  • Developmental time course analyses

  • Layer-specific markers

  • Conditional knockout approaches

• Schwann Cells: When investigating myelin sheath extension:

  • Electron microscopy for ultrastructural analysis

  • Teased fiber preparations

  • Co-localization with myelin markers

For each tissue context, appropriate positive and negative controls must be established, and antibody concentrations may need optimization. Antibody performance in paraffin-embedded tissues has been validated for human kidney samples using citrate buffer (pH 6) for antigen retrieval and AP-staining techniques .

How should I interpret inconsistent results between different applications of PALS1/MPP5 antibodies?

Inconsistent results between different applications may stem from several factors:

• Epitope Accessibility: The PALS1/MPP5 epitope may be masked in certain applications due to:

  • Protein conformational changes in different fixation methods

  • Protein-protein interactions occluding the epitope

  • Post-translational modifications affecting antibody binding

• Application-Specific Considerations:

  • For Western blotting: Denaturing conditions may expose epitopes not accessible in native conformations

  • For immunofluorescence: Fixation and permeabilization protocols significantly affect epitope preservation

  • For flow cytometry: Cell preparation methods influence antibody accessibility to intracellular antigens

• Methodological Approach:

  • Compare your results with published literature

  • Validate findings using multiple antibodies targeting different epitopes

  • Consider using tagged recombinant PALS1/MPP5 as a control

  • Employ knockout/knockdown controls to confirm specificity

• Statistical Analysis: When interpreting quantitative data:

  • Apply appropriate statistical tests (Shapiro-Wilk, t-tests, or non-parametric alternatives)

  • Consider the false discovery rate by applying corrections for multiple testing (e.g., Benjamini-Yekutieli procedure)

  • Use Super-Learner approaches that combine multiple statistical or machine learning models when analyzing complex datasets

If discrepancies persist, contact the antibody manufacturer for technical support and consider whether alternative antibody clones might provide more consistent results across applications.

What quality control measures can detect compromised PALS1/MPP5 antibodies?

Implementing robust quality control measures is essential for detecting compromised PALS1/MPP5 antibodies:

• Positive Control Testing:

  • Use well-characterized tissues or cell lines with known PALS1/MPP5 expression

  • HeLa cells and human kidney tissue are validated positive controls

  • Look for expected subcellular localization patterns (Golgi, cytoplasmic, tight junctions)

• Negative Control Testing:

  • Include unimmunized IgG at the same concentration as your primary antibody

  • Test in tissues or cell lines with minimal PALS1/MPP5 expression

  • For knockout/knockdown validation, compare with wild-type controls

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide

  • A specific antibody should show significantly reduced or abolished signal

• Lot-to-Lot Consistency:

  • Compare performance against previous lots using standardized samples

  • Document and track signal intensity, background levels, and specificity

• Performance Metrics:

  • For quantitative applications, establish detection limits

  • Monitor signal-to-noise ratios over time

  • For peptide ELISA, detection limits of 1:32000 have been established

• Storage and Handling Assessment:

  • Test antibody function after storage at recommended conditions (-20°C)

  • Evaluate performance after multiple freeze-thaw cycles

  • Check for visible precipitation or contamination

If quality control measures suggest antibody compromise, consider requesting a replacement from the manufacturer or switching to an alternative validated antibody clone.

How can I distinguish between specific and non-specific binding in PALS1/MPP5 immunolabeling experiments?

Distinguishing between specific and non-specific binding requires systematic controls and analysis:

• Blocking Optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Optimize blocking time and concentration

  • Include detergents (like Tween-20 or Triton X-100) at appropriate concentrations

• Antibody Titration:

  • Perform serial dilutions to determine optimal antibody concentration

  • Plot signal-to-noise ratio against concentration to identify optimal working dilution

  • Recommended starting concentrations: 10 μg/mL for immunofluorescence, 5 μg/mL for IHC

• Absorption Controls:

  • Pre-incubate primary antibody with purified antigen or immunizing peptide

  • Specific binding should be competitive and show reduced or eliminated signal

• Knockout/Knockdown Validation:

  • Compare labeling in PALS1/MPP5-deficient samples versus wild-type

  • Specific signal should be absent or significantly reduced in knockout samples

• Multiple Antibody Validation:

  • Use antibodies recognizing different epitopes of PALS1/MPP5

  • Consistent labeling patterns across different antibodies suggest specific binding

• Isotype Controls:

  • Use matched isotype controls at the same concentration

  • Unimmunized goat IgG has been used successfully as a negative control

• Cross-Species Reactivity:

  • If studying non-human samples, confirm cross-reactivity or use species-specific antibodies

• Subcellular Localization:

  • Compare observed patterns with known PALS1/MPP5 distribution (Golgi, tight junctions)

  • Co-localization with established markers (e.g., other tight junction proteins) supports specificity

Computational approaches using biophysics-informed models can also help identify specific binding modes when dealing with closely related targets .

How can PALS1/MPP5 antibodies be used to study viral pathogenesis mechanisms?

PALS1/MPP5 antibodies offer powerful tools for investigating viral pathogenesis mechanisms, particularly for viruses that target epithelial barriers:

• SARS-CoV Interaction Studies:

  • PALS1 antibodies can track protein redistribution during infection, as PALS1 moves to the ERGIC/Golgi region where viral E protein accumulates

  • Co-immunoprecipitation assays using PALS1 antibodies can identify viral-host protein interactions, as demonstrated with SARS-CoV E protein

  • Pull-down assays can confirm direct binding between viral proteins and PALS1

• Epithelial Barrier Disruption:

  • PALS1 antibodies can monitor tight junction integrity during viral infection

  • Immunofluorescence studies can reveal how PALS1 sequestration by viral proteins affects junction formation

  • Time-course studies can establish the temporal relationship between PALS1 redistribution and barrier breakdown

• PDZ Domain Interactions:

  • Since the SARS-CoV E protein interacts with PALS1 through a PDZ-binding motif, antibodies specific to this domain can help identify other viral proteins that might employ similar mechanisms

  • Competition assays using PALS1 antibodies can assess therapeutic strategies aimed at preventing these interactions

• 3D Culture Models:

  • PALS1 antibodies can evaluate morphological changes in epithelial cyst formation during infection

  • Immunostaining for PALS1 in organoid cultures can reveal infection-induced polarity defects

• Therapeutic Target Validation:

  • PALS1 antibodies can help validate therapeutic approaches aimed at preventing viral hijacking of this host protein

  • Screening assays can identify compounds that prevent PALS1-viral protein interactions

Research indicates that SARS-CoV E protein acts as a virulence factor by hijacking PALS1, causing severe damage to epithelial barriers . PALS1 antibodies are therefore valuable tools for understanding similar mechanisms in other viral infections that target epithelial barriers.

What advanced computational approaches can optimize PALS1/MPP5 antibody selection for multi-target studies?

Advanced computational approaches can significantly enhance PALS1/MPP5 antibody selection in complex multi-target studies:

• Biophysics-Informed Modeling:

  • Enables identification of different binding modes associated with specific ligands

  • Can disentangle binding modes even for chemically similar ligands

  • Allows prediction of experimental outcomes for new ligand combinations

  • Facilitates generation of novel antibody sequences with customized binding profiles

• Energy Function Optimization:

  • For creating cross-specific antibodies: Jointly minimize energy functions associated with desired ligands

  • For creating highly specific antibodies: Minimize energy for desired targets while maximizing it for undesired targets

  • Mathematical optimization approaches can efficiently navigate complex sequence space

• Statistical Antibody Selection Framework:

  • When dealing with large antibody panels, statistical approaches become essential

  • Data normality can be assessed using the Shapiro-Wilk test

  • For normally distributed data: t-tests can compare mean values between groups

  • For non-normal distributions: finite mixture models can identify latent populations

  • Mann-Wilcoxon tests can be applied for non-parametric comparisons

• Multiple Testing Correction:

  • The Benjamini-Yekutieli procedure under general dependence assumptions can control false discovery rates

  • This approach is particularly valuable when testing many antibodies simultaneously

  • Ensures global false discovery rate of 5% across multiple comparisons

• Super-Learner Predictive Approaches:

  • Combines multiple statistical or machine learning models for antibody performance prediction

  • Can achieve superior AUC values (e.g., 0.801 with 95% CI=0.709-0.892)

  • Particularly valuable when the number of antibody targets exceeds computational feasibility for brute-force approaches (>5 targets)

These computational methods offer solutions to the computational bottlenecks encountered in large-scale antibody selection studies and can identify optimal antibody combinations that might be missed by traditional approaches.

How can cell-type specific PALS1/MPP5 expression patterns inform experimental design?

Understanding cell-type specific PALS1/MPP5 expression patterns is crucial for designing targeted and informative experiments:

• Epithelial Cell Studies:

  • PALS1 is critical for tight junction biogenesis and epithelial polarity establishment

  • Experiments should focus on junction formation, barrier function, and polarity markers

  • Appropriate controls should include polarized epithelial cell lines (e.g., MDCK cells)

  • Look for localization at tight junctions and the apical membrane domain

• Neuronal Development Research:

  • PALS1 maintains cerebellar progenitor cells in an undifferentiated proliferative state

  • Experimental design should incorporate developmental time points

  • Include assays for progenitor proliferation, differentiation, and survival

  • Consider the role of PALS1 in mTOR signaling in neuronal progenitors

• Vascular Endothelium Studies:

  • PALS1 interacts with CDH5 in vascular lumen formation and endothelial membrane polarity

  • Experimental approaches should assess lumen formation and endothelial junction integrity

  • Co-localization with endothelial markers is essential for interpretation

• Schwann Cell Research:

  • PALS1 plays a role in radial and longitudinal extension of myelin sheath

  • Experiments should address myelination processes and Schwann cell-axon interactions

  • Include appropriate myelin markers in co-labeling experiments

• Retinal Development:

  • PALS1 is required for embryonic and postnatal retinal development

  • Experimental design should include developmental stages and retinal layer analysis

  • Consider the interaction of PALS1 with other developmentally regulated proteins

When designing experiments, researchers should:

• Target antibody concentrations based on expected expression levels

• Include appropriate positive and negative controls for each cell type

• Consider conditional knockout/knockdown approaches for cell-type specific analysis

• Choose fixation and antigen retrieval methods optimized for the tissue and cell type of interest

Immunohistochemistry studies have successfully used PALS1 antibodies in human kidney tissue with citrate buffer (pH 6) for antigen retrieval , but parameters may need adjustment for other tissues.