mreg Antibody

What is melanoregulin (MREG) and why is it important in research?

Melanoregulin is a 214-amino acid protein with a molecular weight of 24.9 kDa that functions as a cargo-recognition protein connecting cytoplasmic vesicles to transport machinery . It is primarily expressed in photoreceptor cells and localizes to cytoplasmic vesicles, lysosomes, and cell membranes . MREG has garnered significant research interest due to its role in intracellular transport processes and potential implications in retinal physiology. The protein undergoes post-translational modifications, notably palmitoylation, which likely regulates its membrane association and trafficking functions .

MREG is also known by alternative names including dilute suppressor protein homolog and whn-dependent transcript 2, with evolutionary conservation demonstrated by orthologs in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . This conservation suggests fundamental biological importance across vertebrate lineages, making it a valuable target for comparative studies in cellular biology and disease models.

What are the most common applications for MREG antibodies in research?

MREG antibodies are primarily utilized in the following experimental applications:

When selecting applications, researchers should consider that MREG's subcellular localization in vesicles and membranes makes immunofluorescence particularly valuable for tracking its distribution and trafficking dynamics . Western blotting provides confirmation of antibody specificity and protein size, while more specialized applications like immunoprecipitation can reveal protein-protein interactions that illuminate MREG's functional roles.

How do I select the most appropriate MREG antibody for my research application?

Selection of the optimal MREG antibody requires systematic consideration of several technical parameters:

• Epitope targeting: Determine whether you need antibodies targeting the C-terminal region (as in ABIN2790335) or full-length protein. C-terminal antibodies may be advantageous when studying proteins with conserved functional domains or when terminus-specific detection is required.

• Species reactivity: Verify cross-reactivity with your experimental model. Available antibodies offer diverse reactivity profiles spanning human, mouse, rat, cow, dog, guinea pig, rabbit, and other species . Sequence alignment analysis between species can predict reactivity - for example, some antibodies show 100% predicted reactivity with cow and dog MREG, but only 93% with guinea pig, mouse, and rabbit .

• Clonality considerations:

  • Polyclonal antibodies: Offer broader epitope recognition but potential batch variation

  • Monoclonal antibodies: Provide consistent specificity with potentially narrower epitope recognition

  • Recombinant antibodies: Deliver superior batch-to-batch consistency and sustainable production

• Validation evidence: Prioritize antibodies with experimental validation data specific to your application. Review Western blot images, immunofluorescence patterns, and other validation data provided by manufacturers or in literature .

• Conjugation requirements: Determine whether your application requires unconjugated antibodies or those conjugated to fluorophores (FITC), enzymes (HRP), or affinity tags (biotin) .

The selection process should culminate in obtaining an antibody with demonstrated specificity for MREG in your experimental system and compatibility with your methodological approaches.

What are the optimal protocols for using MREG antibodies in Western blotting experiments?

Western blotting with MREG antibodies requires careful optimization due to the protein's relatively low molecular weight (24.9 kDa) and potential post-translational modifications. A methodological approach includes:

• Sample preparation optimization:

  • Use fresh lysates with protease inhibitors to prevent degradation

  • Include phosphatase inhibitors if studying phosphorylation states

  • Consider membrane-specific extraction protocols given MREG's membrane localization

• Gel electrophoresis parameters:

  • Higher percentage gels (12-15%) provide better resolution for the ~25 kDa MREG protein

  • Consider gradient gels when simultaneously detecting interaction partners

  • Load protein standards that include low molecular weight markers

• Transfer conditions:

  • Use PVDF membranes for better protein retention

  • Optimize transfer time (30-60 minutes) and voltage (80-100V) for this size range

  • Consider semi-dry transfer systems for efficient transfer of smaller proteins

• Blocking and antibody incubation:

  • Test both BSA and milk-based blockers (5% solution)

  • Primary antibody dilutions typically range from 1:500 to 1:2000

  • Optimize incubation time (overnight at 4°C often yields best results)

• Signal detection optimization:

  • Enhanced chemiluminescence (ECL) provides sufficient sensitivity

  • Fluorescent secondary antibodies enable multiplex detection

  • Consider signal enhancement systems for low abundance detection

• Controls and validation:

  • Always include positive controls (tissues/cells known to express MREG)

  • Include negative controls (MREG knockout samples when available)

  • Verify band size corresponds to predicted 24.9 kDa

By following these optimization steps, researchers can achieve specific detection of MREG protein while minimizing background and non-specific signals.

How can I validate the specificity of an MREG antibody for my experimental system?

Comprehensive validation of MREG antibodies requires multi-faceted approaches:

• Genetic validation strategies:

  • MREG knockdown/knockout verification: Test antibody against samples with reduced/absent MREG expression

  • Overexpression systems: Test antibody against samples with increased MREG expression

  • Implement CRISPR-Cas9 editing to generate validation controls

• Biochemical validation approaches:

  • Peptide competition assays: Pre-incubate antibody with immunizing peptide to confirm specificity

  • Molecular weight verification: Confirm detection at expected 24.9 kDa

  • Multiple antibody verification: Test multiple antibodies targeting different epitopes

• Cross-reactivity assessment:

  • Test across multiple species if working with non-human models

  • Verify expected expression patterns in tissues known to express MREG (particularly photoreceptor cells)

  • Implement appropriate negative controls (tissues without MREG expression)

• Application-specific validation:

  • For immunofluorescence: Verify subcellular localization matches known MREG distribution in cytoplasmic vesicles, lysosomes, and cell membranes

  • For Western blotting: Confirm single band at expected molecular weight

  • For immunoprecipitation: Verify enrichment of MREG and known interaction partners

• Documentation standards:

  • Record complete antibody metadata (supplier, catalog number, lot number, dilution)

  • Document all validation experiments with appropriate controls

  • Maintain validation records for reproducibility purposes5

Implementing these validation approaches ensures experimental rigor and addresses the reproducibility concerns frequently associated with antibody-based research5.

What are the best approaches for optimizing immunofluorescence experiments with MREG antibodies?

Optimizing immunofluorescence protocols for MREG detection requires particular attention to preserving subcellular structures given its vesicular and membrane localization:

• Fixation optimization:

  • Compare paraformaldehyde (4%) with methanol fixation

  • Paraformaldehyde better preserves membrane structures

  • Methanol may provide superior epitope accessibility for some antibodies

  • Test fixation durations (10-20 minutes) to minimize artifacts

• Permeabilization parameters:

  • Gentle permeabilization with 0.1-0.2% Triton X-100 for membrane proteins

  • Alternative: 0.1% saponin for reversible permeabilization that better preserves membrane structures

  • Digitonin (50-100 μg/ml) for selective plasma membrane permeabilization

• Blocking optimization:

  • Test normal serum (5-10%) from secondary antibody host species

  • Alternative: 3-5% BSA in PBS

  • Include 0.1% Tween-20 to reduce background

  • Consider addition of 0.3M glycine to reduce aldehyde-induced autofluorescence

• Antibody incubation conditions:

  • Optimize primary antibody dilution (1:100-1:500 range)

  • Test incubation temperature/time combinations (4°C overnight vs. room temperature for 1-2 hours)

  • Include 0.05% Tween-20 in antibody diluent to reduce non-specific binding

• Co-localization strategy:

  • Implement dual labeling with organelle markers:

    • Lysosomal markers (LAMP1, LAMP2)

    • Endosomal markers (EEA1, Rab proteins)

    • Membrane markers (Na+/K+-ATPase)

  • Use high-resolution confocal microscopy for precise localization

• Counterstaining protocol:

  • Nuclear counterstain (DAPI) at 1:1000-1:5000

  • Phalloidin for F-actin visualization to provide cellular context

  • Carefully selected fluorophores to avoid spectral overlap

• Image acquisition parameters:

  • Optimize for MREG's vesicular pattern

  • Use appropriate z-stack sampling for three-dimensional localization

  • Implement deconvolution for improved signal-to-noise ratio

These optimization strategies enable precise visualization of MREG's subcellular distribution, particularly its association with cytoplasmic vesicles, lysosomes, and cell membranes as described in the literature .

How can MREG antibodies be used to investigate protein-protein interactions and trafficking dynamics?

MREG's function as a cargo-recognition protein makes investigation of its interaction network and trafficking dynamics particularly valuable . Advanced methodological approaches include:

• Co-immunoprecipitation (Co-IP) strategies:

  • Use MREG antibodies conjugated to solid supports (protein A/G beads, magnetic beads)

  • Implement crosslinking approaches (DSP, formaldehyde) to capture transient interactions

  • Analyze precipitates by mass spectrometry for unbiased interaction discovery

  • Confirm results with reciprocal Co-IPs using antibodies against identified partners

• Proximity labeling approaches:

  • Generate MREG fusion constructs with BioID or APEX2

  • Express in target cells to biotinylate proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

  • Validate interactions with Co-IP and colocalization studies

• Live-cell imaging techniques:

  • Generate fluorescently tagged MREG constructs (ensure tag doesn't disrupt function)

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to study MREG dynamics

  • Use FRET/FLIM to detect direct protein interactions in living cells

  • Employ particle tracking to follow MREG-positive vesicles

• Super-resolution microscopy applications:

  • STORM or PALM imaging with MREG antibodies to resolve subvesicular localization

  • Dual-color super-resolution to precisely map MREG relative to interaction partners

  • Correlative light-electron microscopy to link molecular specificity with ultrastructural context

• Traffic inhibition experiments:

  • Treat cells with trafficking inhibitors (Brefeldin A, nocodazole, wortmannin)

  • Monitor changes in MREG distribution with immunofluorescence

  • Analyze colocalization with organelle markers under perturbed conditions

  • Assess effects on MREG-dependent cargo transport

These methodologies provide mechanistic insights into MREG's role in coupling cytoplasmic vesicles to transport machinery, illuminating both normal physiological functions and potential pathological disruptions.

What approaches should be employed when encountering discrepancies in MREG antibody-based experimental results?

When researchers encounter contradictory or unexpected results with MREG antibodies, a systematic troubleshooting approach is essential:

• Antibody validation reassessment:

  • Re-verify antibody specificity with knockout/knockdown controls

  • Test alternative antibodies targeting different MREG epitopes

  • Perform peptide competition assays to confirm specificity

  • Check for lot-to-lot variation by obtaining validation data for specific lot5

• Technical parameter investigation:

  • Systematically vary sample preparation methods

  • Test multiple blocking agents to reduce non-specific binding

  • Adjust antibody concentration across a broader range

  • Modify incubation times and temperatures

• Sample-specific considerations:

  • Verify MREG expression in your experimental system

  • Consider potential isoform differences (up to 2 isoforms reported)

  • Assess post-translational modifications that might affect epitope recognition

  • Evaluate species-specific differences if working across multiple models

• Contextual experimental factors:

  • Document cell/tissue culture conditions that might affect MREG expression

  • Control for confluency, passage number, and treatment conditions

  • Consider circadian or cell-cycle dependent expression patterns

  • Standardize lysate preparation to control for extraction efficiency

• Data reconciliation strategies:

  • Implement orthogonal detection methods (mass spectrometry, RNA analysis)

  • Quantitatively compare results across different antibodies and methods

  • Document all experimental variables for transparent reporting

  • Consider biological variability vs. technical artifacts

• Collaborative verification:

  • Engage with other laboratories to independently replicate critical experiments

  • Share detailed protocols to identify potential methodological differences

  • Contribute to community resources like the Only Good Antibodies (OGA) initiative5

This structured approach addresses the reproducibility challenges frequently encountered in antibody-based research while advancing rigorous experimental practices in the field.

How can recombinant and next-generation antibody technologies improve MREG research?

Recent advances in antibody engineering offer significant advantages for MREG research:

• Recombinant antibody advantages:

  • Exceptional batch-to-batch consistency compared to traditional antibodies

  • Defined sequence enables precise epitope targeting

  • Animal-free production supports ethical research practices

  • Sustainable supply without hybridoma dependence

  • Amenable to further engineering and optimization

• Single-domain antibody applications:

  • Smaller size enables access to restricted epitopes

  • Superior penetration in tissues and cells

  • Potential for intracellular expression (intrabodies)

  • Generation of conformation-specific binders for MREG functional states

• Deep learning-based antibody design:

  • Computational generation of antibody sequences with desired properties

  • In-silico optimization for:

    • High expression levels

    • Thermal stability

    • Low hydrophobicity

    • Minimal non-specific binding

  • Accelerated discovery compared to conventional methods

• Multiparametric detection systems:

  • Antibody cocktails targeting multiple MREG epitopes simultaneously

  • Multiplexed detection with differently labeled antibodies

  • Integration with mass cytometry for highly multiplexed analysis

  • Spatial profiling technologies for tissue-level MREG distribution

• Application-optimized formats:

  • Directly conjugated primary antibodies eliminating secondary detection steps

  • Proximity-based detection systems (PLA, CODEX)

  • Antibody fragments optimized for specific applications

  • Bispecific formats targeting MREG and interaction partners simultaneously

• Implementation considerations:

  • Validation requirements remain essential despite technological advances

  • Cost-benefit analysis for transitioning from traditional to recombinant antibodies

  • Integration with established protocols requires optimization

  • Documentation standards for novel reagents

These advanced technologies address many limitations of traditional antibodies while expanding the methodological toolkit available for investigating MREG's biological functions and pathological implications .

What experimental controls are essential when working with MREG antibodies?

Rigorous experimental design with appropriate controls is fundamental to generating reliable data with MREG antibodies:

• Specificity controls:

  • Genetic controls: MREG knockout/knockdown samples

  • Peptide competition: Pre-incubation of antibody with immunizing peptide

  • Isotype controls: Matched isotype antibody at equivalent concentration

  • Secondary-only controls: Omission of primary antibody

• Technical validation controls:

  • Positive controls: Samples known to express MREG (photoreceptor cells)

  • Negative controls: Tissues/cells with minimal MREG expression

  • Dilution series: Demonstration of signal proportionality to protein quantity

  • Multiple antibodies: Independent verification with different MREG antibodies

• Application-specific controls:

  • For Western blotting:

    • Loading controls (β-actin, GAPDH, total protein stains)

    • Molecular weight markers spanning MREG's expected size (24.9 kDa)

    • Reduced/non-reduced sample comparison if relevant

  • For immunofluorescence:

    • Autofluorescence controls (no antibody)

    • Counterstaining for cellular context

    • Colocalization with known MREG-associated structures

    • Z-stack acquisition to confirm three-dimensional distribution

  • For immunoprecipitation:

    • Pre-immune serum controls

    • Non-specific IgG controls

    • Input sample controls

    • Reciprocal IP validation

• Quantitative analysis controls:

  • Standard curves for quantitative applications

  • Technical replicates to assess methodological variation

  • Biological replicates to assess sample variation

  • Statistical power calculations to determine appropriate sample sizes

Implementation of these controls addresses the reproducibility challenges frequently encountered in antibody-based research and provides a framework for rigorous experimental design5.

How should MREG antibody-related methods be documented to ensure experimental reproducibility?

Comprehensive documentation of MREG antibody experiments is essential for reproducibility and follows these methodological principles:

• Antibody metadata documentation:

  • Complete antibody identification (manufacturer, catalog number, lot number)

  • Clonality specification (monoclonal, polyclonal, recombinant)

  • Host species and isotype

  • Epitope information (e.g., C-terminal targeting as in ABIN2790335)

  • RRID (Research Resource Identifier) when available

• Validation documentation:

  • Specific validation performed for the experimental system

  • Images of validation experiments (Western blots, immunofluorescence)

  • Link to repository data if validation was previously published

  • Documentation of controls implemented

• Experimental conditions reporting:

  • Complete buffer compositions with exact pH values

  • Precise antibody dilutions and diluents

  • Incubation times and temperatures

  • Blocking agent composition and concentration

  • Washing procedures (number, duration, buffer composition)

• Sample preparation details:

  • Cell/tissue source with identifier information

  • Growth/maintenance conditions

  • Lysis/extraction procedure with buffer composition

  • Protein quantification method

  • Sample storage conditions

• Image acquisition parameters:

  • Microscope specifications (make, model, objectives)

  • Camera/detector details

  • Acquisition settings (exposure, gain, binning)

  • Software version for image collection

  • Raw image data preservation

• Analysis methodology:

  • Software tools with version numbers

  • Processing steps in sequential order

  • Parameter settings for quantification

  • Statistical methods with justification

  • Data transformation or normalization procedures

This comprehensive documentation approach addresses the transparency issues identified in antibody research reproducibility and aligns with best practices advocated by initiatives like the Only Good Antibodies (OGA) community5.

What methodological approaches can distinguish between MREG isoforms or post-translationally modified variants?

Investigating MREG's various molecular forms requires specialized methodological approaches:

• Isoform-specific detection strategies:

  • Design epitope-specific antibodies targeting unique regions of each isoform

  • Implement RT-PCR with isoform-specific primers as orthogonal validation

  • Use 2D gel electrophoresis to separate isoforms based on both size and charge

  • Apply high-resolution gel systems (gradient gels, Phos-tag) for subtle size differences

• Post-translational modification analysis:

  • For palmitoylation (known MREG modification) :

    • Hydroxylamine treatment to remove palmitate groups

    • Click chemistry approaches with alkyne-palmitate analogs

    • Acyl-biotin exchange (ABE) assay for site-specific analysis

  • For phosphorylation assessment:

    • Phosphatase treatment controls

    • Phos-tag SDS-PAGE for mobility shift detection

    • Phospho-specific antibodies if available

    • Mass spectrometry with phosphopeptide enrichment

• Mass spectrometry approaches:

  • Immunoprecipitate MREG for enrichment prior to MS analysis

  • Apply bottom-up proteomics for sequence coverage and modification mapping

  • Implement top-down proteomics for intact protein analysis of isoforms

  • Use targeted MS methods (PRM, MRM) for quantitative comparison of specific forms

• Cellular distribution analysis:

  • Determine subcellular localization differences between isoforms

  • Track trafficking patterns specific to post-translationally modified forms

  • Implement FRAP or photoactivation to assess mobility differences

  • Use CRISPR-Cas9 to tag endogenous MREG for live-cell imaging

• Functional discrimination methods:

  • Generate isoform-specific knockdowns/knockouts

  • Create non-modifiable mutants (e.g., palmitoylation-deficient)

  • Develop conformation-specific antibodies for functional states

  • Implement proximity labeling to identify isoform-specific interaction partners

These methodological approaches enable researchers to move beyond detection of total MREG protein to understanding the functional significance of its molecular diversity, including the reported two isoforms and post-translational modifications like palmitoylation .

How will AI and computational approaches impact MREG antibody development and application?

Artificial intelligence and computational methods are transforming antibody research with specific implications for MREG studies:

• Deep learning antibody design:

  • Generative Adversarial Networks (GANs) create novel antibody sequences

  • Computational optimization for developability characteristics:

    • High expression levels

    • Thermal stability

    • Low hydrophobicity

    • Minimal non-specific binding

  • Accelerated discovery compared to conventional immunization methods

  • Potential for MREG-specific binders designed entirely in silico

• Structural prediction advancements:

  • AlphaFold2 and RoseTTAFold enable accurate antibody structure prediction

  • Structure-based epitope mapping for optimal MREG targeting

  • Computational docking to predict antibody-MREG interactions

  • Virtual screening of antibody libraries against MREG structural models

• Automated validation pipelines:

  • High-throughput specificity screening across tissue panels

  • Automated image analysis for localizing MREG in cellular contexts

  • Machine learning algorithms for antibody performance prediction

  • Standardized data collection to improve reproducibility5

• Data integration platforms:

  • Aggregation of antibody validation data across laboratories

  • Metadata standardization for enhanced comparability

  • Network analysis of MREG interaction partners

  • Systems biology approaches connecting MREG to broader cellular pathways

• Future methodological developments:

  • Integration with single-cell technologies for heterogeneity assessment

  • Spatial transcriptomics correlation with protein-level detection

  • Computational correction of antibody cross-reactivity

  • Automated experiment design optimization

These technological advances will likely transform MREG research by providing more specific detection tools, enhancing reproducibility, and accelerating discovery of MREG's functional roles in normal physiology and disease contexts 5.

What methodological adaptations are required when studying MREG in disease models or patient samples?

Translational research involving MREG requires specific methodological considerations:

• Clinical sample handling protocols:

  • Optimize fixation for preserved epitopes (particularly important for MREG's membrane localization)

  • Implement antigen retrieval methods suitable for MREG detection

  • Validate antibodies specifically in human tissues

  • Control pre-analytical variables (collection time, processing delay, fixation duration)

• Disease-specific considerations:

  • For retinal degeneration (given MREG's expression in photoreceptor cells) :

    • Layer-specific analysis techniques

    • Correlation with functional visual parameters

    • Comparison with established retinal degeneration markers

    • Control for disease stage and progression

  • For vesicular trafficking disorders:

    • Co-analysis with other trafficking proteins

    • Assessment of cargo accumulation

    • Ultrastructural analysis of vesicle morphology

    • Functional trafficking assays in patient-derived cells

• Patient-derived materials methodology:

  • Standardized protocols for tissue biobanking

  • Establishment of patient-derived cell models

  • iPSC differentiation protocols preserving MREG expression

  • Correlation of MREG alterations with clinical parameters

• Quantitative assessment approaches:

  • Digital pathology for standardized quantification

  • Multiplex immunofluorescence for contextual analysis

  • Mass cytometry for high-dimensional profiling

  • Spatial proteomics for tissue microenvironment analysis

• Methodological validation requirements:

  • Verify antibody performance in disease-altered microenvironments

  • Control for treatment effects on epitope accessibility

  • Implement blocking of endogenous peroxidases/biotin in clinical samples

  • Include appropriate disease and normal control tissues

These methodological adaptations ensure reliable MREG detection and quantification in complex disease contexts while maintaining scientific rigor and reproducibility in translational research applications.

How can researchers contribute to improving standardization and reproducibility in MREG antibody applications?

Researchers can actively advance standardization efforts through these methodological approaches:

• Validation data contribution:

  • Implement comprehensive validation protocols for each application

  • Share detailed validation data through repositories

  • Report negative results regarding antibody performance

  • Contribute to community resources like the Only Good Antibodies (OGA) initiative5

• Methods standardization practices:

  • Adopt standardized reporting formats for antibody experiments

  • Include complete antibody metadata in publications

  • Participate in multi-laboratory validation studies

  • Implement minimum information guidelines for antibody experiments

• Resource development and sharing:

  • Generate and share MREG knockout/knockdown validation controls

  • Develop recombinant MREG standards for quantitative applications

  • Create and distribute isoform-specific detection tools

  • Establish reference materials for inter-laboratory standardization

• Collaborative research networks:

  • Participate in ring trials for antibody performance assessment

  • Engage in direct laboratory exchanges to harmonize protocols

  • Contribute to consensus guidelines for MREG detection

  • Support repositories for antibody characterization data

• Training and education initiatives:

  • Implement rigorous training in antibody validation methodologies

  • Develop verification procedures for new laboratory members

  • Share troubleshooting protocols and expertise

  • Promote open science practices in antibody research

• Publication and reporting practices:

  • Include detailed methods sections with complete protocol information

  • Provide thorough antibody validation data in publications or supplements

  • Report batch/lot numbers and sources

  • Describe all control experiments performed

By implementing these practices, researchers contribute to addressing the reproducibility challenges in antibody-based research while advancing the standardization necessary for consistent and reliable MREG detection across laboratories5.