Mpdz Antibody

What is MPDZ and why is it significant in research?

MPDZ (also known as MUPP1, multiple PDZ domain protein, multi-PDZ domain protein 1, and HYC2) is a scaffolding protein with significant research importance. In humans, the canonical protein consists of 2070 amino acid residues with a molecular mass of 221.6 kDa and localizes primarily to the cell membrane . It is expressed in multiple tissues including heart, brain, placenta, liver, skeletal muscle, kidney, and pancreas . MPDZ functions as a member of the NMDAR signaling complex and may play critical roles in AMPAR potentiation and synaptic plasticity in excitatory synapses . Its evolutionary conservation across species (mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken) indicates fundamental biological importance . Recent research has also demonstrated MPDZ's interaction with proteins like DAPLE, which together regulate apical cell constriction, suggesting roles in epithelial morphogenesis .

What are the common applications for MPDZ antibodies in research?

MPDZ antibodies are utilized in numerous research applications, with Western Blot (WB) and Immunohistochemistry (IHC) being the most widely employed techniques . These antibodies enable the detection, localization, and quantification of MPDZ protein in various experimental contexts. Specifically:

• Western Blot (WB): Used for detecting MPDZ protein in tissue or cell lysates, typically at the expected molecular weight of approximately 221.6 kDa

• Immunohistochemistry (IHC): Applied to visualize the distribution and localization of MPDZ in tissue sections

• Immunofluorescence (IF): Employed to examine subcellular localization, particularly at cell-cell boundaries in the apical domain

• Co-Immunoprecipitation (co-IP): Used to study protein-protein interactions, such as the physical association between MPDZ and DAPLE

Recommended dilutions for optimal results typically range from 1:50-1:200 for IHC and 1:200-1:2000 for WB applications .

How should MPDZ antibodies be stored and handled to maintain activity?

Proper storage and handling of MPDZ antibodies are crucial for maintaining antibody activity and experimental reproducibility. Based on manufacturer recommendations:

• Long-term storage: Store at -20°C for up to one year

• Short-term storage: For frequent use, store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles as they can compromise antibody integrity and binding efficiency

• Storage form: Most commercial MPDZ antibodies are supplied in buffered aqueous glycerol solutions, which helps maintain stability

When working with the antibodies, always follow good laboratory practices including proper aliquoting upon first thaw to minimize freeze-thaw cycles, use of clean pipette tips, and maintenance of sterile conditions to prevent contamination.

How can researchers validate the specificity of MPDZ antibodies?

Antibody validation is essential for ensuring experimental reliability. For MPDZ antibodies, consider these validation approaches:

• Positive control tissues: Use tissues known to express MPDZ (brain, heart, kidney, liver, etc.)

• Negative controls: Include samples without primary antibody and samples from MPDZ-knockout models

• Blocking peptide experiments: Preincubate the antibody with immunogen peptide to confirm specificity

• Multiple antibody validation: Use antibodies targeting different epitopes of MPDZ

• Molecular weight confirmation: Verify that the detected band appears at the expected size (~221.6 kDa)

• Cross-reactivity testing: Check whether the antibody recognizes MPDZ across different species as claimed by manufacturers

Thorough validation across multiple techniques (WB, IHC, ICC-IF) ensures reliable experimental outcomes and interpretable results.

What factors should be considered when selecting an MPDZ antibody for specific applications?

When selecting an MPDZ antibody, researchers should carefully evaluate several factors:

Whenever possible, select antibodies that have been validated through enhanced validation protocols that test for specificity, reproducibility, and appropriate target recognition under experimental conditions similar to your own.

How should researchers optimize immunostaining protocols for MPDZ detection in different tissue types?

Optimizing immunostaining protocols for MPDZ requires tissue-specific considerations:

• Fixation method selection:

  • For membrane-associated MPDZ: 4% paraformaldehyde is generally effective

  • For preserved morphology: Consider short glutaraldehyde fixation (0.1-0.5%)

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) works for many tissues

  • For highly fixed tissues, try enzyme digestion or alternative buffers (EDTA pH 8.0)

• Blocking parameters:

  • Use 5-10% serum from secondary antibody host species

  • Add 0.1-0.3% Triton X-100 for intracellular epitopes

  • Consider adding 1% BSA to reduce background

• Antibody dilution optimization:

  • Start with manufacturer's recommended range (typically 1:50-1:200 for IHC)

  • Perform titration experiments to determine optimal signal-to-noise ratio

  • Extend primary antibody incubation (overnight at 4°C) for weak signals

• Tissue-specific considerations:

  • For brain tissue: Longer fixation periods may require stronger antigen retrieval

  • For epithelial cells: Preserve junction integrity with milder permeabilization

  • When examining apical cell constriction: Consider using EpH4 epithelial cells as a model system

Creating a systematic optimization matrix with these variables will help identify ideal conditions for each specific tissue and application.

What are the best approaches for dual-labeling experiments involving MPDZ and its interaction partners?

Dual-labeling experiments are crucial for studying MPDZ interactions with partners like DAPLE. Consider these approaches:

• Antibody selection strategy:

  • Choose primary antibodies from different host species (e.g., rabbit anti-MPDZ with mouse anti-DAPLE)

  • If same-species antibodies must be used, consider directly conjugated antibodies or sequential immunostaining protocols

• Optimized co-staining protocol:

  • Perform individual single-staining controls alongside dual-staining

  • Block with mixed sera from both secondary antibody host species

  • Include absorption controls to rule out cross-reactivity

• MPDZ-DAPLE co-localization analysis:

  • Use confocal microscopy with appropriate controls for spectral bleed-through

  • Apply quantitative co-localization analysis (Pearson's correlation, Manders' overlap coefficient)

  • Focus on cell-cell boundaries of the apical cell domain where co-localization has been observed

• Proximity ligation assay (PLA) approach:

  • Consider PLA to detect protein-protein interactions within 40nm

  • Use species-specific PLA probes compatible with your primary antibodies

  • Optimize fixation to preserve both protein epitopes

• Co-immunoprecipitation validation:

  • Follow protocols similar to those used to demonstrate MPDZ-DAPLE interaction

  • Include appropriate controls (IgG control, single transfection controls)

  • Validate results with reciprocal co-IP (IP with anti-DAPLE, blot for MPDZ)

These approaches provide complementary evidence for protein interactions and overcome technical limitations of individual methods.

How can researchers address non-specific binding issues with MPDZ antibodies?

Non-specific binding can complicate interpretation of MPDZ antibody results. These strategies can help minimize such issues:

• Optimize blocking conditions:

  • Increase blocking agent concentration (5-10% normal serum)

  • Add 0.1-0.5% non-ionic detergents (Tween-20, Triton X-100) to reduce hydrophobic interactions

  • Consider specialized blocking reagents (protein-free blockers, commercial blockers)

• Antibody dilution adjustment:

  • Increase antibody dilution incrementally to reduce non-specific binding

  • Compare signal at multiple dilutions (1:50, 1:100, 1:200, 1:500, 1:1000)

  • Balance sensitivity with specificity

• Buffer and wash optimization:

  • Add 0.05-0.1% Tween-20 to all wash buffers

  • Increase salt concentration (150-500mM NaCl) in wash buffers

  • Extend washing times and increase wash frequency

• Pre-absorption strategy:

  • Pre-absorb antibody with tissue powder from non-expressing tissue

  • For polyclonal antibodies, consider affinity purification against the immunogen

• Technical controls:

  • Include no-primary-antibody controls

  • Use isotype controls at the same concentration

  • Compare with another validated MPDZ antibody targeting a different epitope

When persistent non-specific binding occurs, systematic evaluation of these parameters will help identify the optimal conditions for your specific experimental system.

What approaches should be taken when Western blot results for MPDZ show unexpected band patterns?

MPDZ Western blots may show complex band patterns due to its large size, multiple isoforms, and potential post-translational modifications. When facing unexpected patterns:

• Analyze band size discrepancies:

  • Expected full-length MPDZ: ~221.6 kDa

  • Lower molecular weight bands may represent:

    • Alternative splice variants/isoforms (up to 4 reported)

    • Proteolytic fragments (use fresh samples with protease inhibitors)

    • Cross-reactive proteins (validate with knockout/knockdown controls)

• Sample preparation optimization:

  • For high molecular weight proteins like MPDZ:

    • Use lower percentage gels (6-8%)

    • Extend transfer time or use specialized transfer methods for large proteins

    • Consider gradient gels for better resolution

  • Ensure complete protein denaturation (extend boiling time, increase SDS concentration)

• Validation experiments:

  • Peptide competition to confirm specificity of each band

  • siRNA/shRNA knockdown to verify band reduction

  • Comparison with recombinant protein standards

  • Test multiple antibodies targeting different MPDZ epitopes

• Technical considerations:

  • For multiple bands, consider:

    • Post-translational modifications (phosphorylation, glycosylation)

    • Tissue-specific expression patterns of isoforms

    • Protein degradation during sample preparation

Creating a detailed record of band patterns across different tissues, sample preparation methods, and antibody lots will help establish expected patterns for your experimental system.

How can immunoprecipitation protocols be optimized for studying MPDZ interactions?

Immunoprecipitation (IP) of MPDZ requires careful optimization due to its size and membrane association. Consider these strategies:

• Lysis buffer optimization:

  • For membrane-associated MPDZ:

    • Use buffers containing 0.5-1% NP-40 or Triton X-100

    • Consider adding 0.1-0.25% SDS to increase solubilization

    • Include appropriate protease and phosphatase inhibitors

  • Buffer composition should preserve protein-protein interactions while effectively extracting MPDZ

• Antibody selection and coupling:

  • Choose antibodies validated for IP applications

  • Consider covalent coupling to beads to avoid antibody contamination in eluates

  • Pre-clear lysates with beads alone to reduce non-specific binding

• IP protocol refinements:

  • For MPDZ-DAPLE co-IP:

    • Follow validated protocols that demonstrated this interaction

    • Use epitope-tagged constructs (MYC-DAPLE, FLAG-MPDZ) for efficient pull-down

    • Include appropriate controls (β-catenin, integrin β1) to confirm specificity

• Analysis of complexes:

  • For large proteins like MPDZ (221.6 kDa):

    • Use gradient gels for better resolution

    • Consider specialized electrophoresis conditions for high molecular weight proteins

    • Silver staining for sensitive detection of interaction partners

• Validation approaches:

  • Perform reciprocal IP (IP with anti-DAPLE, blot for MPDZ)

  • Include negative controls (IgG, irrelevant antibody)

  • Confirm with alternative methods (proximity ligation assay, FRET)

These optimizations will help ensure successful isolation of MPDZ and its interaction partners while minimizing artifacts and non-specific interactions.

How can MPDZ antibodies be utilized to investigate its role in synaptic plasticity and NMDAR signaling?

MPDZ's involvement in NMDAR signaling complexes and synaptic plasticity can be investigated through several advanced approaches using specific antibodies:

• Subcellular localization studies:

  • Use super-resolution microscopy techniques (STORM, PALM, SIM) with MPDZ antibodies

  • Perform co-localization studies with NMDAR subunits, AMPAR components, and other PSD proteins

  • Quantify changes in localization following synaptic activity or plasticity-inducing stimuli

• Activity-dependent modifications:

  • Employ phospho-specific antibodies to track MPDZ post-translational modifications

  • Combine with synaptic fractionation to assess redistribution following NMDAR activation

  • Correlate modifications with electrophysiological measurements of synaptic strength

• In situ proximity analysis:

  • Apply proximity ligation assays to detect MPDZ interactions with NMDAR subunits

  • Quantify interaction changes following LTP or LTD induction

  • Combine with electrophysiological recordings in the same preparations

• Functional intervention studies:

  • Use antibodies for acute disruption of MPDZ interactions in electrophysiology experiments

  • Compare results with genetic approaches (CRISPR/Cas9, conditional knockouts)

  • Validate specificity with rescue experiments using antibody-resistant MPDZ constructs

• Circuit-level analysis:

  • Employ immunohistochemistry to map MPDZ expression across different neuronal populations

  • Correlate with electrophysiological properties and plasticity capabilities

  • Examine changes in disease models associated with synaptic dysfunction

These approaches provide complementary insights into MPDZ's dynamic role in synaptic function and plasticity mechanisms.

What methodological approaches can assess MPDZ's role in cellular polarity and junction formation?

MPDZ's localization at cell-cell boundaries of the apical domain suggests important roles in cellular polarity and junction formation. These methodological approaches can assess these functions:

• Advanced imaging strategies:

  • Employ live-cell imaging with fluorescent protein-tagged MPDZ

  • Combine with MPDZ antibody staining in fixed cells to validate tag function

  • Use FRAP (Fluorescence Recovery After Photobleaching) to assess dynamic exchange at junctions

• Functional perturbation approaches:

  • Compare MPDZ knockdown/knockout with antibody blocking experiments

  • Assess junction formation kinetics using calcium-switch assays

  • Measure epithelial barrier function through TEER (Trans-Epithelial Electrical Resistance)

• 3D culture systems:

  • Utilize MPDZ antibodies in immunostaining of epithelial cysts and organoids

  • Assess polarity establishment in 3D vs. 2D systems

  • Examine co-localization with other polarity complexes (Par, Crumbs, Scribble)

• Mechanical force measurements:

  • For apical constriction studies:

    • Use traction force microscopy combined with MPDZ immunostaining

    • Apply MPDZ antibodies in epithelial cell systems like EpH4

    • Correlate MPDZ localization with actomyosin contractility markers

• Junction remodeling analysis:

  • Track MPDZ dynamics during junction remodeling events

  • Assess phosphorylation status during junction assembly/disassembly

  • Investigate MPDZ-DAPLE cooperation in regulating apical constriction

These approaches provide mechanistic insights into MPDZ's functions in establishing and maintaining cell polarity and junction integrity.

How can researchers use MPDZ antibodies to investigate its involvement in pathological conditions?

MPDZ's diverse functions suggest potential roles in various pathological conditions. Researchers can use MPDZ antibodies to investigate these aspects:

• Expression analysis in disease tissues:

  • Compare MPDZ expression levels and localization patterns in normal vs. pathological tissues

  • Employ tissue microarrays with MPDZ antibodies for high-throughput screening

  • Correlate expression with disease progression markers and patient outcomes

• Cell-type specific pathology assessment:

  • Use multi-label immunofluorescence to identify affected cell populations

  • Combine with cell-type specific markers in complex tissues

  • Quantify alterations in subcellular distribution in disease states

• Functional interaction studies in disease models:

  • Investigate alterations in MPDZ-DAPLE interactions in epithelial pathologies

  • Assess NMDAR complex composition changes in neurological disorders

  • Examine junction integrity in barrier dysfunction disorders

• Post-translational modification analysis:

  • Develop or obtain phospho-specific MPDZ antibodies

  • Compare modification patterns between normal and pathological conditions

  • Correlate with activity of relevant kinases and phosphatases

• Therapeutic targeting assessment:

  • Use antibodies to validate target engagement in drug development

  • Monitor MPDZ complex formation following therapeutic intervention

  • Assess restoration of normal localization patterns after treatment

These approaches enable systematic investigation of MPDZ's potential contributions to disease mechanisms and evaluation of therapeutic strategies targeting MPDZ-dependent pathways.

How might new antibody technologies enhance MPDZ research applications?

Emerging antibody technologies offer exciting possibilities for advancing MPDZ research:

• Single-domain antibodies (nanobodies):

  • Smaller size allows better penetration into tissue and access to sterically hindered epitopes

  • Potential for intrabody applications to track MPDZ in living cells

  • Higher specificity for particular conformational states or protein complexes

• Recombinant antibody fragments:

  • Fab and scFv formats with defined specificity and reduced background

  • Potential for site-specific conjugation with minimal impact on binding

  • Compatible with phage display selection for novel epitope targeting

• Antibody-based proximity sensors:

  • Split fluorescent protein complementation systems coupled to anti-MPDZ antibodies

  • FRET-based biosensors to detect MPDZ conformational changes or interactions

  • Kinase activity reporters to monitor MPDZ-associated signaling events

• Spatially-resolved antibody applications:

  • Integration with spatial transcriptomics for correlative multi-omics

  • Highly multiplexed imaging using cycling immunofluorescence or mass cytometry

  • Super-resolution microscopy compatible antibody formats

• Engineered specificity antibodies:

  • Isoform-specific antibodies targeting unique regions of MPDZ variants

  • Conformation-specific antibodies recognizing active vs. inactive states

  • Modification-specific antibodies detecting particular post-translational modifications

These technologies will expand the toolkit available for investigating MPDZ biology with greater precision, specificity, and spatiotemporal resolution.

What considerations are important when integrating MPDZ antibody data with other -omics approaches?

Integration of antibody-based MPDZ data with other -omics approaches requires careful consideration:

• Correlation with transcriptomics:

  • Account for potential discrepancies between mRNA and protein levels

  • Consider time-lag between transcriptional changes and protein abundance

  • Validate antibody specificity when expression patterns differ from transcriptomics data

• Proteomics integration:

  • Compare antibody-based quantification with mass spectrometry data

  • Consider epitope masking due to protein interactions or modifications

  • Use multiple antibodies targeting different epitopes to validate proteomics findings

• Phosphoproteomics correlation:

  • Use phospho-specific antibodies to validate phosphoproteomics identification

  • Consider how phosphorylation might affect antibody binding

  • Integrate temporal dynamics of phosphorylation with functional outcomes

• Interactomics validation:

  • Use co-IP with MPDZ antibodies to validate interaction partners identified in interactomics

  • Consider context-specificity of interactions (cell type, stimulation conditions)

  • Apply proximity ligation assays to confirm key interactions in situ

• Data integration frameworks:

  • Develop standardized workflows for normalizing and comparing antibody-based data with other -omics

  • Account for differences in dynamic range and sensitivity

  • Implement appropriate statistical approaches for integrative analysis

These considerations ensure proper interpretation of multi-modal data and leverage complementary strengths of different methodologies for comprehensive understanding of MPDZ biology.