MPZL2 Antibody

What is MPZL2 and why is it significant in research?

MPZL2 (myelin protein zero-like 2) is a type 1 transmembrane protein of the myelin P0 protein family that mediates homophilic cell-cell adhesion. In humans, the canonical protein has 215 amino acid residues with a molecular weight of 24.5 kDa . MPZL2 contains an Ig-like V-type (immunoglobulin-like) domain and two potential glycosylation sites . It is also known by several synonyms including EVA, EVA1, epithelial V-like antigen 1, and DFNB111 .

MPZL2 has gained significant research interest due to its association with autosomal recessive nonsyndromic hearing impairment. Studies have identified homozygous truncating variants of MPZL2 (such as c.72delA) in families with progressive sensorineural hearing loss . The protein's role in cell adhesion and its expression patterns in the inner ear make it a critical target for studying mechanisms of hearing loss and potential therapeutic interventions.

What types of MPZL2 antibodies are available for research applications?

Several types of MPZL2 antibodies are available for research applications, primarily:

• Polyclonal antibodies: These recognize multiple epitopes on the MPZL2 protein. For example, Proteintech offers a rabbit polyclonal antibody (11787-1-AP) that targets the full MPZL2 protein .

• Region-specific antibodies: Some antibodies target specific regions of MPZL2, such as the N-terminal region (e.g., Aviva Systems Biology's ARP46654_P050) .

• Host species variation: MPZL2 antibodies are produced in different host species, including rabbit and mouse, with varying reactivity profiles .

These antibodies are validated for multiple applications, including Western Blot (1:1000-1:4000 dilution), Immunohistochemistry, Immunofluorescence, ELISA, and Flow Cytometry, with demonstrated reactivity against human, mouse, and rat samples .

How can I validate the specificity of an MPZL2 antibody for my research?

Validating antibody specificity is crucial for reliable research outcomes. For MPZL2 antibodies, consider these methodological approaches:

• Knockout/knockdown validation: The gold standard for antibody validation is testing in knockout or knockdown models. Several published studies have used MPZL2 knockout models to confirm antibody specificity . Analysis of cochleae from Mpzl2-mutant mice has been effectively used to confirm the specificity of anti-MPZL2 antibodies in immunohistochemistry applications .

• Multiple detection methods: Use complementary techniques like Western blot alongside immunofluorescence or immunohistochemistry. For Western blot validation, expected molecular weight for MPZL2 is 24-30 kDa, which accounts for potential post-translational modifications like glycosylation .

• Cell line validation: Test antibodies in cell lines with known MPZL2 expression. A431 and HaCaT cells have been documented as positive controls for MPZL2 detection in Western blot applications .

• Peptide competition assays: For antibodies derived from synthetic peptide immunogens (like the KLH-conjugated synthetic peptide from amino acids 67-94 of the central region of human MPZL2) , conduct peptide competition assays to verify epitope specificity.

What are the optimal conditions for using MPZL2 antibodies in immunofluorescence studies of inner ear tissues?

For immunofluorescence studies of MPZL2 in inner ear tissues, the following protocol has been successfully employed:

• Tissue preparation:

  • Dissect tympanic bullae containing the cochleae from mice (P0 stage recommended)

  • Locally perfuse with 4% PFA through round and oval windows

  • Fix overnight at 4°C in 4% PFA

  • Rinse in 1X PBS

• Immunostaining procedure:

  • Permeabilize whole mount cochlea with 0.5% Triton X-100

  • Block in 5% BSA for 1 hour at room temperature

  • Incubate with primary anti-MPZL2 antibody (e.g., Proteintech 11787-1-AP) overnight at 4°C

  • For co-staining, anti-Occludin antibody can be used alongside MPZL2 antibody

  • Alexa Fluor 647-Phalloidin is effective for co-detection of F-actin

• Imaging considerations:

  • Confocal microscopy (e.g., Zeiss LSM710) provides optimal resolution for visualizing MPZL2 localization

  • Pay particular attention to subcellular localization, as MPZL2 demonstrates asymmetric distribution in hair cells

This approach has successfully revealed MPZL2 expression in mouse inner ear, particularly in auditory inner and outer hair cells .

How does MPZL2 expression differ across developmental stages and tissue types, and what implications does this have for antibody selection?

MPZL2 demonstrates dynamic expression patterns across developmental stages and tissues, requiring careful consideration for antibody selection:

• Developmental expression:

  • MPZL2 expression has been detected in fetal tissues, including cochlea as early as 8 weeks of gestation

  • In the inner ear, expression patterns change between neonatal (P4) and adult (P28) stages, with protein primarily detected in outer hair cells (OHCs), Deiters' cells (DCs), and at contacts between DCs and the basilar membrane

• Tissue-specific expression:

  • MPZL2 is widely expressed across many tissue types

  • Expression has been documented in thymus epithelium with downregulation during thymocyte developmental progression

  • In adult mice, expression has been confirmed in lung, liver, kidney, brain, and cochlea at P15

• Implications for antibody selection:

  • For developmental studies, ensure antibodies recognize conserved regions that are present throughout development

  • For cross-species studies, consider antibodies validated across multiple species (human, mouse, rat)

  • For studies focusing on post-translational modifications, select antibodies that can detect glycosylated forms, as MPZL2 undergoes glycosylation

Understanding these expression patterns is crucial for experimental design and interpretation, particularly in studies involving hearing loss mechanisms, as MPZL2 plays a tissue-specific role in the inner ear despite its broader expression pattern .

What approaches should be used to investigate MPZL2 function in cell adhesion within the inner ear epithelium?

Investigating MPZL2's function in cell adhesion within the inner ear epithelium requires multiple complementary approaches:

• Gene expression analysis:

  • RNA-seq of cochlear tissues from wild-type and MPZL2-deficient models has revealed significant changes in genes related to cell adhesion, extracellular matrix (ECM) organization, and basement membrane integrity

  • Key differentially expressed genes include ECM components (COL9A1/2/3, EMILIN1, POSTN, TNXB, and LAMA1/2)

  • Quantitative RT-PCR validation of these targets provides a mechanistic framework for understanding MPZL2's role in adhesion

• Protein-protein interaction studies:

  • Co-immunoprecipitation using anti-MPZL2 antibodies to identify binding partners

  • Investigation of homophilic interactions, as MPZL2 mediates cell-cell adhesion through homophilic binding

  • Examination of interactions with other junctional proteins such as ZO-1 and Occludin, which have been co-stained with MPZL2 in cochlear studies

• Functional assays in mouse models:

  • Analysis of auditory brainstem responses (ABRs) in MPZL2-deficient models shows progressive hearing loss, more pronounced at high frequencies

  • Histological analysis of cochlear tissues reveals altered organization of outer hair cells and supporting cells, along with degeneration of the organ of Corti

  • Immunofluorescence microscopy using anti-MPZL2 antibodies reveals asymmetric subcellular localization in hair cells, suggesting specialized functions at specific cellular junctions

• CRISPR-based approaches:

  • Development of humanized mouse models (like hMPZL2 Q74X/Q74X) that recapitulate human MPZL2 deafness mutations provides valuable systems for studying protein function and testing potential therapeutics

  • Base editing approaches targeting disease-causing mutations (such as c.220C>T) show promise for rescuing hearing function and restoring inner ear structural integrity

What are common sources of false positives/negatives when using MPZL2 antibodies, and how can they be addressed?

When working with MPZL2 antibodies, several factors can lead to false results:

• Sources of false positives:

  • Cross-reactivity with other Myelin P0 protein family members due to sequence homology

  • Non-specific binding in glycoprotein-rich tissues, as MPZL2 is glycosylated

  • Secondary antibody cross-reactivity, particularly in multi-color immunofluorescence studies

  Solutions:

  • Include appropriate blocking steps (5% BSA has been effective)

  • Use antibodies validated against knockout/knockdown models

  • Perform peptide competition assays to confirm specificity

  • Include isotype controls to identify non-specific binding

• Sources of false negatives:

  • Epitope masking due to protein glycosylation or other post-translational modifications

  • Insufficient antigen retrieval, especially in fixed tissues

  • Protein degradation during sample preparation

  Solutions:

  • Optimize antigen retrieval methods for fixed tissues

  • Use multiple antibodies targeting different epitopes of MPZL2

  • Consider detection of MPZL2 mRNA (via RT-PCR) alongside protein detection to confirm expression

  • Verify storage conditions (store at -20°C, avoid repeated freeze/thaw cycles)

• Sample-specific considerations:

  • In cochlear tissues, autofluorescence can interfere with signal detection

  • The small size of inner ear structures requires careful microdissection and processing

  Solutions:

  • Include appropriate autofluorescence controls

  • Optimize fixation time to preserve epitopes while maintaining tissue architecture (overnight fixation in 4% PFA at 4°C has been effective)

  • Consider tissue-specific dilutions (starting with 1:1000 for Western blot applications)

How can I optimize detection of MPZL2 in Western blot applications when working with inner ear samples?

Optimizing Western blot detection of MPZL2 in inner ear samples requires special considerations due to the tissue's limited size and complex composition:

• Sample preparation:

  • Pool multiple cochleae to obtain sufficient protein (typically 4-6 mouse cochleae)

  • Use efficient lysis buffers containing appropriate protease inhibitors

  • Consider ultrasonication to ensure complete membrane protein extraction, as MPZL2 is a transmembrane protein

  • Avoid harsh detergents that might disrupt protein epitopes

• Electrophoresis and transfer optimization:

  • Use gradient gels (4-20%) to optimize separation of the 24-30 kDa protein

  • Include positive control samples (A431 or HaCaT cells have shown consistent MPZL2 expression)

  • Consider wet transfer methods for more efficient transfer of membrane proteins

  • Use PVDF membranes for better protein retention

• Antibody incubation parameters:

  • Start with recommended dilutions (1:1000-1:4000) and optimize based on signal strength

  • Extended primary antibody incubation (overnight at 4°C) often improves detection

  • Include proper controls (tissue from Mpzl2-knockout or mutant mice) in the same blot

  • Be aware that glycosylated forms may appear at slightly higher molecular weights (up to 30 kDa)

• Detection system considerations:

  • Enhanced chemiluminescence (ECL) systems provide good sensitivity

  • For samples with low MPZL2 expression, consider more sensitive detection methods like femto-ECL substrates

  • Fluorescent secondary antibodies can provide better quantitative analysis and lower background

• Multiplex strategies:

  • Consider co-detection with markers of specific inner ear cell types

  • Include loading controls appropriate for inner ear tissues (β-actin or GAPDH)

  • Run parallel gels to analyze both glycosylated and deglycosylated forms if studying post-translational modifications

How can MPZL2 antibodies be utilized in studying the molecular mechanisms of hereditary hearing loss?

MPZL2 antibodies serve as valuable tools for investigating hereditary hearing loss mechanisms:

• Genotype-phenotype correlation studies:

  • MPZL2 antibodies enable protein-level validation of genetic findings in families with DFNB111 hearing loss

  • Immunohistochemistry with anti-MPZL2 antibodies can reveal specific cellular defects in cochlear tissues from individuals or models with MPZL2 mutations

  • Studies have revealed that MPZL2 deficiency affects high-frequency hearing more severely, correlating with protein expression patterns in the basal turn of the cochlea

• Cellular mechanism investigations:

  • MPZL2 antibodies have revealed the protein's localization in outer hair cells, Deiters' cells, and at contacts with the basilar membrane

  • Immunofluorescence studies using these antibodies have demonstrated that MPZL2 deficiency disrupts the organization of outer hair cells and supporting cells

  • Co-staining with markers for cell junctions (ZO-1, Occludin) and ion channels (KCNQ1, Kir4.1) has helped elucidate MPZL2's role in maintaining cochlear epithelium integrity

• Pathway analysis:

  • Antibody-based protein analysis, combined with transcriptomic studies, has revealed that MPZL2 deficiency affects genes involved in cell adhesion, extracellular matrix organization, and basement membrane integrity

  • This approach has identified potential downstream effectors including collagens (COL9A1/2/3), laminins (LAMA1/2), and other adhesion molecules

• Therapeutic development:

  • MPZL2 antibodies provide essential tools for validating gene therapy approaches, such as adeno-associated virus (AAV)-delivered base editing

  • In humanized mouse models (hMPZL2 Q74X/Q74X), antibodies can confirm restoration of protein expression following genetic interventions

  • The efficacy of therapies can be assessed at both the protein level (via antibody detection) and functional level (via auditory brainstem responses)

What novel approaches could leverage MPZL2 antibodies for investigating cell adhesion defects in diseases beyond hearing loss?

While MPZL2 has been primarily studied in the context of hearing loss, its broader expression pattern and role in cell adhesion suggest potential applications in other diseases:

• Thymic development and immunological disorders:

  • MPZL2 is expressed in thymus epithelium and regulated during thymocyte developmental progression

  • Antibody-based studies could investigate MPZL2's role in thymic epithelial cell interactions and potential contributions to immunological disorders

  • Flow cytometry applications using anti-MPZL2 antibodies could help characterize thymic cell populations and their interactions

• Epithelial integrity in other tissues:

  • As a mediator of homophilic cell-cell adhesion, MPZL2 may play roles in maintaining epithelial barriers in multiple organs

  • Antibody-based tissue screening could identify previously unrecognized sites of functional importance

  • Co-localization studies with other junctional proteins could reveal tissue-specific adhesion complexes

• Cancer research applications:

  • Cell adhesion molecules often play dual roles in cancer progression and suppression

  • Immunohistochemistry panels including anti-MPZL2 antibodies could assess expression changes in epithelial cancers

  • Such studies might reveal correlations between MPZL2 expression patterns and tumor invasiveness or metastatic potential

• Regenerative medicine:

  • Understanding MPZL2's role in maintaining specialized epithelia could inform tissue engineering approaches

  • Antibody-based monitoring of MPZL2 expression during differentiation of stem cells into epithelial lineages might provide insights into optimizing cell-cell interactions

  • For inner ear regeneration specifically, MPZL2 antibodies could help assess the structural integrity of engineered cochlear tissues

• Comparative and evolutionary studies:

  • MPZL2 orthologs have been reported in multiple species including mouse, rat, bovine, chimpanzee, and chicken

  • Cross-reactive antibodies could facilitate comparative studies of epithelial junctions across species

  • Such studies might reveal evolutionary adaptations in cell adhesion mechanisms relevant to tissue-specific functions

These approaches could expand our understanding of MPZL2's biological functions beyond hearing and potentially identify new therapeutic targets for diseases involving epithelial dysfunction.

What are the key considerations for using MPZL2 antibodies in high-resolution microscopy techniques for inner ear research?

High-resolution microscopy of MPZL2 in inner ear tissues presents unique challenges and opportunities:

• Sample preparation considerations:

  • The complex 3D architecture of the organ of Corti requires specialized preparation techniques

  • For whole mount preparations, careful microdissection is essential to preserve cochlear structures while allowing antibody access

  • The protocol of perfusing 4% PFA through round and oval windows, followed by overnight fixation at 4°C, preserves both structure and antigenicity

  • Permeabilization with 0.5% Triton X-100 and blocking with 5% BSA has proven effective for reducing background while maintaining specific signals

• Advanced microscopy techniques:

  • Confocal microscopy (e.g., Zeiss LSM710) provides excellent resolution for visualizing MPZL2 localization within the organ of Corti

  • Super-resolution techniques (STED, STORM, SIM) could further resolve MPZL2's subcellular distribution, particularly at cellular junctions

  • For thick specimens, two-photon microscopy may provide better depth penetration while reducing photobleaching

  • Light-sheet microscopy offers advantages for studying intact cochlear samples with minimal photodamage

• Multi-channel imaging strategies:

  • Co-staining with F-actin (using Alexa Fluor 647-Phalloidin) provides structural context for MPZL2 localization

  • Including markers for specific cell types (Myosin VIIa for hair cells, SOX2 for supporting cells) helps identify cell-specific expression patterns

  • Junction markers (ZO-1, Occludin) can reveal co-localization with MPZL2 at cell-cell contacts

  • Ion channel markers (KCNQ1, Kir4.1) provide functional context for MPZL2 localization

• Quantitative analysis approaches:

  • Develop consistent imaging parameters across specimens to allow quantitative comparisons

  • Consider fluorescence intensity, distribution patterns, and co-localization coefficients

  • Z-stack imaging is essential for capturing the full 3D architecture of MPZL2 distribution

  • Age-matched comparisons are critical, as MPZL2 distribution changes during development and with progressive hearing loss

How can RNA and protein analyses be combined to provide comprehensive insights into MPZL2 expression and function?

Integrating RNA and protein analyses creates a more complete understanding of MPZL2 biology:

• Complementary expression analyses:

  • RT-PCR has been used to verify MPZL2/Mpzl2 transcript expression across tissues (lung, liver, kidney, brain, cochlea) and developmental stages (embryonic day 17.5, postnatal day 15)

  • Combine with Western blot or immunohistochemistry using anti-MPZL2 antibodies to correlate transcript and protein levels

  • This approach has revealed that the c.220C>T mutation causes nonsense-mediated mRNA decay rather than producing a truncated protein

• Transcriptome-proteome integration:

  • RNA-seq of cochlear tissues from wild-type and MPZL2-deficient models has identified differentially expressed genes related to cell adhesion and ECM organization

  • Protein-level validation of these findings using antibodies against MPZL2 and its potential interactors provides functional context

  • Key differentially expressed genes (COL9A1/2/3, EMILIN1, IBSP, POSTN, TNXB, LAMA1/2) can be validated at both RNA (by qRT-PCR) and protein levels (by immunodetection)

• Mutation impact assessment:

  • For truncating mutations (c.72delA, c.220C>T), antibodies can determine whether truncated proteins are produced or if nonsense-mediated decay eliminates the transcript

  • RT-PCR analysis of splice site mutations can reveal altered splicing events, while antibodies can detect resulting protein variants

  • This combined approach confirmed that in Mpzl2-mutant mice with deletion of exons 2-3, transcript splicing from exon 1 to exon 4 occurs, but protein expression is absent

• Temporal and spatial correlation:

  • In situ hybridization for MPZL2 mRNA combined with immunohistochemistry using anti-MPZL2 antibodies can reveal whether transcript and protein distributions match

  • This approach can identify post-transcriptional regulation mechanisms and protein trafficking patterns

  • For developmental studies, tracking both mRNA and protein expression provides insights into the timing of gene activation and protein accumulation in specific structures