MPZL3 Antibody

What is MPZL3 and why is it significant in research?

MPZL3 (Myelin protein zero-like protein 3) is a member of the myelin P0 protein family. The human MPZL3 gene is located at chromosome 11q23.3 and encodes a 235-amino acid polypeptide with an Immunoglobulin V-type (IgV) domain . MPZL3 functions as a membrane adhesion protein and may play important roles in immune function. The significance of MPZL3 was initially discovered through studies of the rough coat (rc) mutation in mice, where a missense mutation in the MPZL3 gene leads to severe skin and hair abnormalities, including cyclic and progressive hair loss and sebaceous gland hypertrophy . This discovery highlighted MPZL3's potential importance in epithelial biology and has spurred further research into its functions in various tissues.

What are the typical applications for MPZL3 antibodies in research?

MPZL3 antibodies are primarily used in Western Blot (WB) and ELISA applications . These applications allow researchers to detect and quantify MPZL3 protein expression in various tissues and experimental conditions. The antibody has been validated for reactivity with both human and mouse samples , making it versatile for comparative studies across species. Beyond these standard applications, researchers have also employed MPZL3 antibodies for indirect immunofluorescence to determine protein localization in tissue sections, particularly in skin where MPZL3 expression has been detected in keratinocytes of the epidermis, hair follicles, and sebocytes .

What is the expected molecular weight of MPZL3 in Western blot analysis?

When working with MPZL3 antibodies, researchers should be aware of the potential discrepancy between calculated and observed molecular weights:

The 28 kDa band likely represents the endogenous MPZL3 of unmodified form, while the 70 kDa band may represent a post-translationally modified form of the protein . In some experimental systems, researchers have also detected bands of higher molecular weight (~80 and ~110 kDa) which could represent different post-translational modifications or protein products from different transcripts . When analyzing experimental results, it's important to consider these multiple forms of the protein.

How should MPZL3 antibody be stored to maintain its efficacy?

Proper storage of MPZL3 antibody is crucial for maintaining its reactivity and specificity. The recommended storage conditions are:

Following these storage recommendations will help ensure the antibody remains effective for the expected duration . Repeated freeze-thaw cycles should be avoided as they may compromise antibody quality.

What controls should be included when validating MPZL3 antibody specificity?

When validating MPZL3 antibody specificity, several controls should be considered:

• Peptide competition assay: Preabsorbing the antibody with the immunizing peptide (such as DKLTIDWTYRPPSSSRT) at increasing molar ratios (1:3, 1:10, 1:32) should progressively reduce or eliminate specific binding .

• Expression system controls: Testing the antibody against cells transfected with an expression vector encoding MPZL3 (with or without an epitope tag) compared to non-transfected cells can confirm specificity .

• Comparison with another detection method: When possible, validating results with another antibody or detection technique enhances confidence in specificity.

• Negative controls: Including negative controls such as normal serum instead of primary antibody in immunofluorescence experiments is essential to distinguish specific from non-specific staining .

Such rigorous validation ensures that experimental findings truly reflect MPZL3 biology rather than artifacts of cross-reactivity.

What is the tissue distribution pattern of MPZL3 expression?

MPZL3 displays a broad tissue distribution pattern as determined by RT-PCR analysis. The gene is expressed in multiple organs with particularly high levels detected in:

• Brain

• Heart

• Liver

• Skin

Other organs also express MPZL3, albeit at potentially lower levels . Western blot analysis of adult mouse organs has detected a single band of approximately 70 kDa across various tissues examined . This widespread expression pattern suggests that MPZL3 may have diverse physiological roles beyond those initially identified in skin. Researchers investigating MPZL3 function should consider this broad expression profile when designing experiments and interpreting results.

How is MPZL3 protein localized within cells and tissues?

Indirect immunofluorescence studies have revealed that MPZL3 protein is predominantly localized to the plasma membrane of cells, consistent with its predicted function as a transmembrane protein involved in cell adhesion . More specifically:

• In the skin, MPZL3 is expressed in keratinocytes of the epidermis and hair follicles

• Strong staining is observed around the plasma membrane

• Cytoplasmic staining is also detected, but nuclear staining is absent

• In mice with hypertrophic sebaceous glands (rc/rc mice), MPZL3 protein expression is also detected in sebocytes

This localization pattern supports the hypothesis that MPZL3 functions as a cell adhesion molecule, similar to other members of the myelin P0 protein family. The membrane localization is consistent with the protein's predicted structure, which includes a signal peptide, an Ig-like V-type domain, and a transmembrane domain .

Does MPZL3 expression vary in different pathological conditions?

While the search results don't provide comprehensive information on MPZL3 expression in various pathological conditions, they do highlight the phenotype associated with MPZL3 mutation in the rough coat (rc) mice. These mice develop severe skin and hair abnormalities, including cyclic and progressive hair loss and sebaceous gland hypertrophy . Despite the mutation (Arg100→Gln substitution), MPZL3 protein distribution patterns appear similar between normal and rc/rc mouse skin . This suggests that the mutation may affect protein function rather than expression or localization.

Researchers investigating MPZL3 in disease contexts should consider examining both expression levels and functional aspects of the protein, as pathological effects might result from altered function rather than merely changed expression patterns.

How do alternative splicing variants of the MPZL3 gene affect protein function and antibody recognition?

MPZL3 gene exhibits alternative splicing that generates at least two transcripts:

• A six-exon transcript encoding a 237-amino acid protein (full-length MPZL3)

• A two-exon transcript encoding a shorter 96-amino acid polypeptide

Both transcripts show similar tissue distribution patterns . The shorter polypeptide possesses a signal peptide but has only a portion of the Ig-like domain and lacks the transmembrane domain. This structural difference likely affects its subcellular localization and function compared to the full-length protein.

When using MPZL3 antibodies, researchers should consider which isoforms will be recognized based on the antibody's epitope. The antibody described in the search results (25513-1-AP) was raised against amino acids 32-235 of human MPZL3 , suggesting it would recognize the full-length protein but might not detect all splice variants. Additionally, EST evidence suggests the existence of other MPZL3 transcript forms , adding another layer of complexity to antibody selection and experimental design.

What is the relationship between MPZL3 and other members of the myelin P0 protein family?

MPZL3 shows significant homology with other members of the myelin P0 protein family, particularly:

• MPZ (Myelin Protein Zero): Within the Ig V-type domain, murine MPZL3 shares 40.0% identity and 54.2% similarity at the amino acid level with murine MPZ.

• MPZL2/EVA1 (Epithelial V-like Antigen 1): Within the Ig V-type domain, murine MPZL3 shares 36.1% identity and 60.5% similarity at the amino acid level with murine EVA1.

All consensus residues within the Ig V-type domain, including the cysteines, the N-glycosylation site, and the arginine corresponding to Arg100 in murine MPZL3 (the residue mutated in rc/rc mice), are conserved between these three proteins .

Interestingly, the Mpzl3 gene is located approximately 1 kb downstream of the Eva1 gene, and exons 2 and 3 are identical in size between the two genes (167 and 211 bp, respectively), suggesting that one of these genes might have arisen through tandem duplication . This close evolutionary relationship may have implications for functional redundancy or compensation in experimental knockdown or knockout studies.

What approaches can be used to study MPZL3 post-translational modifications?

The significant discrepancy between calculated (26 kDa) and observed (70 kDa, 28 kDa) molecular weights of MPZL3 strongly suggests extensive post-translational modifications . To study these modifications, researchers might consider the following approaches:

• Enzymatic deglycosylation: Treatment with glycosidases (PNGase F, Endo H, O-glycosidase) can remove N- and O-linked glycans, potentially revealing which bands represent glycosylated forms of MPZL3.

• Phosphatase treatment: To assess phosphorylation status, samples can be treated with phosphatases prior to Western blotting.

• Immunoprecipitation followed by mass spectrometry: This approach can identify specific modifications and their sites within the protein.

• Site-directed mutagenesis: Mutating potential modification sites (glycosylation, phosphorylation, etc.) and observing changes in apparent molecular weight can confirm the nature and location of modifications.

• Subcellular fractionation: Different modified forms may localize to different cellular compartments; fractionation followed by Western blotting can reveal such patterns.

Understanding these modifications is crucial as they likely influence MPZL3's function, stability, localization, and interactions with other proteins.

What are common issues encountered when using MPZL3 antibodies and how can they be resolved?

When working with MPZL3 antibodies, researchers may encounter several technical challenges:

• Multiple bands in Western blots: As MPZL3 can appear at different molecular weights (28 kDa, 70 kDa, and potentially higher) , distinguishing specific signal from non-specific bands can be challenging. Solution: Use positive controls (tissues known to express MPZL3 like brain, liver, or skin) and conduct peptide competition assays to confirm specificity.

• Weak or no signal: This could result from low MPZL3 expression or suboptimal antibody conditions. Solution: Optimize protein extraction methods, increase antibody concentration, extend incubation times, or try enhanced chemiluminescence detection systems.

• High background: Non-specific binding can obscure specific signals. Solution: Increase blocking time/concentration, optimize washing steps, decrease antibody concentration, or try different blocking agents.

• Inconsistent results between experiments: This could result from antibody degradation or variation in experimental conditions. Solution: Use consistent protocols, prepare fresh working solutions, and store antibodies according to manufacturer recommendations.

• Discrepancies between transcript and protein levels: Post-transcriptional regulation may cause discrepancies. Solution: Use multiple detection methods to confirm results and consider investigating post-transcriptional mechanisms.

How can MPZL3 antibodies be validated for immunofluorescence applications?

Validating MPZL3 antibodies for immunofluorescence requires a systematic approach:

• Positive and negative tissue controls: Include tissues known to express MPZL3 (e.g., skin, brain, liver) and tissues with minimal expression as controls.

• Peptide competition: Preincubate the antibody with the immunizing peptide before staining to confirm specificity. Specific staining should be abolished or significantly reduced .

• Secondary antibody-only controls: Omit primary antibody to identify potential non-specific binding of secondary antibodies.

• Comparison with in situ hybridization: When possible, compare protein localization (immunofluorescence) with mRNA expression patterns.

• Knockout or knockdown validation: If available, tissues from MPZL3 knockout animals or cells with MPZL3 knockdown provide stringent specificity controls.

• Cross-validation with multiple antibodies: Using antibodies targeting different epitopes can confirm localization patterns.

The search results indicate successful validation of immunofluorescence using MPZL3 antibodies in skin sections, revealing expression in keratinocytes of the epidermis and hair follicles, with prominent plasma membrane localization .

What considerations should be made when designing experiments to study MPZL3 function?

When designing experiments to study MPZL3 function, researchers should consider:

• Expression patterns: MPZL3 is expressed in multiple tissues including brain, heart, liver, and skin . Experimental design should account for this broad expression and potential tissue-specific functions.

• Alternative splicing: The presence of multiple transcript variants (at least a six-exon and a two-exon form) necessitates careful consideration of which isoforms are targeted in functional studies.

• Potential redundancy: MPZL3's homology to MPZ and MPZL2/EVA1 suggests possible functional redundancy. Compensatory mechanisms may mask phenotypes in single-gene manipulation studies.

• Post-translational modifications: The discrepancy between calculated and observed molecular weights indicates substantial post-translational modifications that may be functionally significant and require specific analytical approaches.

• Subcellular localization: The plasma membrane localization of MPZL3 suggests functions in cell-cell adhesion or signaling. Experimental readouts should include appropriate measures of these processes.

• Species differences: While MPZL3 shows conservation across species, there may be species-specific differences in expression, regulation, or function. The antibody shows reactivity with both human and mouse samples , facilitating comparative studies.

• Control selection: Given the technical challenges in MPZL3 detection, rigorous controls should be implemented in all experiments to ensure validity of findings.

How has our understanding of MPZL3 evolved through recent research?

While the search results primarily focus on foundational studies of MPZL3, including its identification as the gene mutated in rough coat (rc) mice , they highlight several key advances in our understanding:

• Identification of MPZL3 as a cell adhesion molecule: Sequence analysis and structural predictions have placed MPZL3 in the myelin P0 protein family, suggesting function in cell adhesion .

• Characterization of tissue expression patterns: MPZL3 expression has been demonstrated in multiple tissues, with high levels in brain, heart, liver, and skin .

• Subcellular localization: Immunofluorescence studies have revealed predominantly plasma membrane localization of MPZL3 in keratinocytes and sebocytes .

• Recognition of multiple protein forms: Western blot analyses have identified multiple forms of MPZL3 protein, suggesting extensive post-translational modifications .

• Development of research tools: Specific antibodies have been generated and validated for detection of MPZL3 in various applications including Western blotting and immunofluorescence .

These advances provide a foundation for future studies investigating MPZL3's specific functions in various physiological and pathological contexts.

What are potential applications of MPZL3 antibodies in investigating skin disorders?

Given MPZL3's expression in skin and the skin phenotype observed in rc/rc mice , MPZL3 antibodies hold significant potential for investigating various skin disorders:

• Sebaceous gland disorders: The hypertrophy of sebaceous glands in rc/rc mice suggests MPZL3's involvement in sebaceous gland regulation. MPZL3 antibodies could be used to examine expression changes in conditions like acne, sebaceous hyperplasia, or sebaceous adenoma.

• Hair loss disorders: The progressive hair loss in rc/rc mice indicates MPZL3's role in hair follicle maintenance. Immunohistochemical analysis using MPZL3 antibodies might reveal altered expression or localization in alopecia areata, androgenetic alopecia, or other hair loss conditions.

• Epidermal differentiation disorders: MPZL3's expression in epidermal keratinocytes suggests potential involvement in epidermal differentiation. MPZL3 antibodies could be valuable for investigating conditions like psoriasis, atopic dermatitis, or ichthyosis.

• Wound healing: As a cell adhesion molecule expressed in skin, MPZL3 might play roles in wound healing processes. MPZL3 antibodies could help track expression changes during various stages of wound healing.

• Skin cancer: Alterations in cell adhesion molecules often accompany malignant transformation. MPZL3 antibodies might be useful in examining potential changes in expression or localization in skin cancers like basal cell carcinoma, squamous cell carcinoma, or melanoma.

Research in these areas would benefit from the availability of specific MPZL3 antibodies validated for applications like immunohistochemistry, flow cytometry, and protein interaction studies.

What emerging technologies might enhance MPZL3 research in the future?

Several emerging technologies could significantly advance MPZL3 research:

• CRISPR/Cas9-mediated genome editing: Precise modification of the MPZL3 gene could generate new model systems for functional studies, including knockout models, knock-in models with specific mutations (like the rc mutation), or models with tagged endogenous MPZL3 for localization and interaction studies.

• Single-cell transcriptomics: This approach could reveal cell type-specific expression patterns of MPZL3 and its splice variants across tissues, providing insights into potential cell-specific functions.

• Spatial transcriptomics and proteomics: These technologies could map MPZL3 expression with spatial resolution in tissues, potentially revealing microenvironmental regulation of expression.

• Proximity labeling approaches: Techniques like BioID or APEX could identify proteins that interact with MPZL3 in living cells, helping elucidate its functional networks.

• Super-resolution microscopy: Advanced imaging techniques could provide detailed information about MPZL3's subcellular localization and potential co-localization with other proteins.

• High-throughput screening: Screening approaches could identify small molecules or biologics that modulate MPZL3 function, potentially leading to therapeutic applications.

• Organoid and tissue-on-chip technologies: These systems could facilitate studies of MPZL3 function in more physiologically relevant contexts than traditional cell culture.

Incorporation of these technologies into MPZL3 research could accelerate understanding of its biological functions and potential roles in disease.