MPP5 Antibody

What is MPP5 and why is it an important research target?

MPP5 (Membrane Protein Palmitoylated 5), also known as PALS1 (Protein Associated with Lin Seven 1), is a highly conserved apical complex protein essential for cell polarity, fate, and survival. It plays critical roles in:

• Neurogenesis and brain development

• Establishment and maintenance of cell polarity in epithelial cells

• Tight junction and adherens junction biogenesis

• Retinal development and morphogenesis

• Schwann cell myelination

• Cerebellar histogenesis and cortical development

Recent research has linked rare de novo heterozygous variants in MPP5 to global developmental delay (GDD), intellectual disability (ID), language delay/regression, and behavioral changes . The protein is also essential for maintaining progenitor pools required for neurogenesis, with depletion leading to conditions such as microcephaly .

How should I select an appropriate MPP5 antibody for my research application?

Selection of an appropriate MPP5 antibody depends on several critical factors:

• Research application: Different applications require antibodies with specific properties:

  • For Western blot: Both polyclonal and monoclonal antibodies can work well

  • For immunohistochemistry: Consider antibodies validated for this application

  • For flow cytometry: Select antibodies specifically tested for flow applications

• Species reactivity: Ensure compatibility with your experimental model:

  • Human MPP5 research: Multiple validated options available

  • Mouse/rat models: Select antibodies with confirmed cross-reactivity

• Epitope region: Consider the protein domain you need to target:

  • N-terminal antibodies: Useful for detecting full-length MPP5

  • Domain-specific antibodies: When studying specific functional regions

• Validation status: Prioritize antibodies validated through:

  • Knockout/knockdown controls

  • Multiple applications

  • Citation in peer-reviewed publications

What are the optimal protocols for detecting MPP5 by Western blot?

Western blot detection of MPP5 requires specific optimization steps for reliable results:

Sample Preparation:

• Use appropriate lysis buffers containing protease inhibitors

• For brain tissue samples, specialized extraction protocols improve yield

• Human fibroblast samples require 15 μg of protein for reliable detection

Electrophoresis and Transfer Parameters:

• Use reducing conditions with SDS-PAGE

• MPP5 appears at approximately 75-80 kDa on western blots

• For optimal transfer, use PVDF membranes rather than nitrocellulose

Antibody Incubation:

• Primary antibody concentration: 0.5-1 μg/mL shows good results

• Primary incubation: Overnight at 4°C yields better signal-to-noise ratio

• Secondary antibody: HRP-conjugated anti-species IgG at 1:2000-1:5000 dilution

Controls and Normalization:

• GAPDH (36 kDa) serves as an effective loading control

• Normalization using fluorescence-based systems (e.g., LI-COR Odyssey) enables accurate quantification

The Western blot protocol from the neurogenesis study demonstrated a 35% reduction in MPP5 protein expression in cortical lysates of heterozygous conditional knockout animals and similar reduction in cultured fibroblasts derived from patients with nonsense variants .

How should I optimize immunofluorescence protocols for MPP5 localization studies?

Optimizing immunofluorescence for MPP5 localization requires attention to several technical aspects:

Fixation and Permeabilization:

• Paraformaldehyde fixation (4%) for 10-15 minutes preserves MPP5 localization

• Permeabilization with 0.15% Triton X-100 enables antibody access while maintaining structural integrity

Antibody Concentration and Incubation:

• Primary antibody: 5-10 μg/mL shows optimal results for most MPP5 antibodies

• Incubation time: 1-2 hours at room temperature or overnight at 4°C

• Secondary antibody: Alexa Fluor conjugates at 1-2 μg/mL provide good signal

Signal Localization and Expected Patterns:

• Epithelial cells: Tight junctions and apical membrane domains

• Neurons: Cell body and axons, particularly in Purkinje cells

• HeLa cells: Golgi apparatus and cytoplasmic staining

• Retinal cells: Outer limiting membrane junctions

Recommended Controls:

• Negative control: Species-matched non-immune IgG at equivalent concentration

• Peptide competition: Pre-incubation with immunizing peptide

• DAPI nuclear counterstain for structural context

A key consideration for MPP5 localization studies is that redistribution of the protein from cell junctions to cytoplasmic compartments may occur in certain pathological conditions or experimental manipulations affecting cell polarity .

How can MPP5 antibodies be used to investigate neurogenesis defects in developmental disorders?

MPP5 antibodies have proven instrumental in investigating neurogenesis defects associated with developmental disorders through several methodological approaches:

Comparative Expression Analysis:

• Western blot quantification can detect haploinsufficiency in patient samples

• Studies have shown ~35% reduction in MPP5 protein in patient-derived fibroblasts

• Comparison between wild-type and heterozygous MPP5 conditional knockout models reveals dose-dependent effects

Localization Studies in Developing Neural Tissue:

• Immunofluorescence can track MPP5 distribution at the apical surface of ventricular zone

• Co-staining with apical complex markers (Crumbs proteins, Par3) reveals altered localization in MPP5-deficient models

• Time-course studies during peak neurogenesis (E14.5 in mice) capture critical periods of MPP5 function

Functional Consequences Assessment:

• Analyze progenitor pool maintenance using co-staining with proliferation markers

• Assess apical junction formation and ventricular surface integrity

• Measure changes in cortical thickness and architecture in MPP5-deficient models

Methodological Example from Research:
Research using conditional knockout mouse models demonstrated that MPP5 depletion leads to:

• Decreased Crumbs protein localization at the ventricular surface

• Reduction of Par3 at apical junctions

• Displacement of apical complex proteins to basal regions

• Increased apoptotic cell death reducing progenitor populations and neuron numbers

These findings establish MPP5 antibodies as valuable tools for investigating the molecular mechanisms underlying neurodevelopmental disorders associated with MPP5 mutations.

What approaches can be used to study MPP5's role in hematopoietic stem cell differentiation?

Recent research has identified MPP5 as a significant component in hematopoietic stem cell (HSC) differentiation, particularly in multipotent progenitor (MPP) populations. The following methodological approaches using MPP5 antibodies are effective:

Flow Cytometry Analysis:

• MPP5 antibodies (10 μg/mL) can be used for intracellular staining of fixed/permeabilized cells

• Combined with surface markers (CD34, CD135, CD48, CD150) to identify specific progenitor populations

• Applications include phenotyping MPP5-expressing cells within the LSK (Lin⁻Sca-1⁺c-Kit⁺) compartment

Single-Cell Analysis Integration:

• Flow sorting based on MPP5 expression followed by scRNA-seq

• Computational integration with surface marker data (scADT-seq) enables refined classification

• This approach has identified MPP5 as functionally located between HSCs and more committed progenitors

Functional Characterization Methods:

• Transplantation studies comparing MPP5⁺ and MPP5⁻ populations

• Colony formation assays to assess lineage potential

• Long-term culture systems to evaluate self-renewal capacity

Research Finding Example:
A comprehensive study demonstrated that mouse CD34⁺MPP5 (LSK CD135⁻CD48⁻CD150⁻) represents a distinct multipotent progenitor population that:

• Supports initial emergency myelopoiesis upon transplantation

• Provides stable long-term lymphoid reconstitution

• Can generate MPP1-4 but not HSCs

• Is located immediately downstream of HSCs in differentiation trajectories

This research approach established a single-cell framework that identified MPP5 as functionally and molecularly distinct from other HSPC populations, providing new insights into hematopoietic hierarchy.

How can I address non-specific binding issues with MPP5 antibodies?

Non-specific binding can significantly impact the interpretation of MPP5 antibody results. The following methodological approaches can help address these issues:

Antibody Validation Strategies:

• Confirm antibody specificity using MPP5 knockout/knockdown controls

• Perform peptide competition assays using the immunizing peptide

• Test multiple antibodies targeting different MPP5 epitopes

Western Blot Optimization:

• Increase blocking time/concentration (5% BSA or milk for 1-2 hours)

• Adjust antibody concentration (typically 0.5-1 μg/mL is optimal)

• Include 0.1-0.2% Tween-20 in wash buffers

• Use gradient gels to better resolve the 75-80 kDa MPP5 band from potential non-specific bands

Immunofluorescence Protocol Refinement:

• Implement more stringent blocking with normal serum (5-10%) from secondary antibody species

• Increase washing steps (5-6 washes of 5 minutes each)

• Use fluorophore-conjugated Fab fragments instead of whole IgG secondary antibodies

• Include DAPI counterstain to better contextualize subcellular localization

Flow Cytometry Considerations:

• Implement Fluorescence Minus One (FMO) controls

• Test fixation protocols (paraformaldehyde fixed HeLa cells with 0.5% Triton showed good results)

• Use species-matched IgG control at identical concentration

Systematic Validation Example:
Research demonstrated that flow cytometric analysis of paraformaldehyde-fixed HeLa cells (permeabilized with 0.5% Triton) using MPP5 antibody at 10 μg/mL followed by Alexa Fluor 488 secondary antibody (1 μg/mL) produced specific staining when compared to unimmunized goat IgG control .

What quality control measures should be implemented when using MPP5 antibodies in multiplex assays?

When incorporating MPP5 antibodies into multiplex assays (co-immunoprecipitation, multi-color immunofluorescence, CyTOF, etc.), specific quality control measures ensure reliable results:

Cross-Reactivity Prevention:

• Test antibody combinations individually before multiplexing

• Select MPP5 antibodies raised in different host species than other target antibodies

• Use highly cross-adsorbed secondary antibodies

• Consider directly conjugated primary antibodies to eliminate secondary antibody cross-reactivity

Signal Separation Validation:

• Include single-stain controls for each fluorophore/channel

• Perform proper compensation when using flow cytometry or spectral imaging

• Test for bleed-through between channels using spectral unmixing software

• Include samples stained with all antibodies except MPP5 to confirm signal specificity

Quantitative Controls:

• Use calibration beads appropriate for your detection system

• Include biological samples with known MPP5 expression levels

• Implement standardized gating strategies for flow cytometry applications

• Apply consistent thresholding in image analysis workflows

Co-localization Analysis Quality Control:

• Perform point spread function calibration before co-localization studies

• Use appropriate statistical methods (Pearson's coefficient, Manders' overlap coefficient)

• Validate co-localization with orthogonal biochemical methods

Research Application Example:
Studies examining MPP5 interaction with apical polarity complex proteins (Crumbs, Par3) demonstrated that immunofluorescence co-staining required careful optimization. Researchers found that sequentially applying primary antibodies (rather than co-incubation) and using highly cross-adsorbed secondary antibodies minimized artificial co-localization signals and improved detection of genuine protein-protein interactions at the ventricular surface of neural progenitors .

How should I interpret changes in MPP5 localization versus expression level in disease models?

Distinguishing between altered MPP5 localization and expression level changes provides critical mechanistic insights in disease models. Consider these methodological approaches:

Combined Localization and Expression Assessment:

• Perform parallel Western blot (expression level) and immunofluorescence (localization) analyses

• Use subcellular fractionation to quantify MPP5 distribution across cellular compartments

• Implement live cell imaging with fluorescently-tagged MPP5 to track dynamic redistribution

Quantitative Localization Analysis Methods:

• Apply line scan intensity profiles across cellular regions (e.g., apical-basal axis)

• Perform quantitative co-localization analysis with compartment markers

• Use high-content imaging systems for population-level quantification of localization patterns

Distinguishing Patterns in Neuronal Models:

• Expression reduction without localization change: Suggests haploinsufficiency mechanism

• Mislocalization with normal expression: Indicates trafficking/interaction defects

• Reduction specifically at apical junctions: Points to polarity complex assembly issues

Research Finding Context:
In studies of MPP5 heterozygous conditional knockout mice, researchers observed both a 35% reduction in total MPP5 expression and a profound decrease in apical surface localization . This was accompanied by decreased localization of interacting proteins (Crumbs, Par3) at the ventricular surface with displacement to basal regions, suggesting that both reduced expression and mislocalization contribute to the observed neurogenesis defects .

What are the considerations for using MPP5 antibodies in clinical diagnostic research?

While MPP5 antibodies are powerful research tools, their potential application in clinical diagnostic research requires specific methodological considerations:

Reproducibility and Standardization Requirements:

• Select antibodies with demonstrated lot-to-lot consistency

• Establish standardized staining protocols with detailed SOPs

• Implement automated staining platforms when possible

• Develop quantitative scoring systems for interpretation

Tissue-Specific Optimization:

• For neural tissue: Antigen retrieval optimization is critical (citrate buffer pH 6 has shown good results for paraffin sections)

• For peripheral blood samples: Specific fixation and permeabilization protocols for intracellular flow cytometry

• For patient-derived fibroblasts: Standardized culture conditions before analysis

Correlation with Molecular Testing:

• Combine MPP5 immunostaining with genetic testing for MPP5 variants

• Develop immunoassays that can distinguish wild-type from mutant MPP5 proteins

• Correlate MPP5 expression/localization patterns with specific mutation types

Clinical-Research Translation Challenges:

• Limited validation in large clinical cohorts

• Need for reference ranges across developmental stages and tissues

• Current limitation to research use only (RUO) applications

• Requirements for analytical and clinical validation before diagnostic use

Research-Clinical Gap Analysis:
Studies have identified rare de novo heterozygous variants in MPP5 in patients with global developmental delay, language issues, and behavioral changes . While immunostaining can detect protein expression changes in patient fibroblasts, significant work remains to establish standardized diagnostic protocols.

Currently, MPP5 antibodies should be considered investigational tools for clinical research rather than validated diagnostic reagents, with emphasis on their value in understanding disease mechanisms rather than establishing clinical diagnoses.