Metrnl Antibody

What is Metrnl protein and why is it significant in research?

Metrnl is a secreted protein (approximately 30 kDa) with emerging functions in immune regulation, metabolism, and tissue repair. Research significance stems from its roles in:

• Modulating inflammatory responses by promoting M2 macrophage polarization

• Inducing adipose thermogenesis through Type 2 immune cascade activation

• Involvement in various pathological conditions including autoimmune diseases, inflammatory skin disorders, and metabolic dysfunction

Structurally, Metrnl contains an N-terminal signal peptide (45 amino acids) and lacks transmembrane regions, resulting in a mature secreted protein of 266 amino acids . Western blot analysis typically reveals bands between 34-43 kDa, heavier than calculated due to post-translational modifications .

Which experimental applications are most suitable for Metrnl antibodies?

Metrnl antibodies demonstrate utility across multiple applications with varying optimization requirements:

For optimal results, validation in your specific experimental system is essential, particularly regarding species reactivity and antibody format .

How are Metrnl antibodies validated for research authenticity?

Scientific validation typically involves multiple complementary approaches:

• Specificity confirmation: Testing against recombinant Metrnl protein in direct ELISA

• Cross-reactivity assessment: Evaluation against multiple species (human, mouse, rat)

• Expression pattern verification: Comparison with established tissue distribution patterns, including adipose tissue, intestinal epithelial cells, and neuronal cells

• Knockout controls: Validation using Metrnl-KO models to confirm specificity

• Molecular weight verification: Confirming expected band sizes (typically 34-43 kDa for glycosylated forms)

How should researchers select appropriate Metrnl antibodies for specific applications?

Selection criteria should prioritize:

• Target species concordance: Ensure the antibody recognizes your species of interest (human, mouse, rat)

• Application validation: Verify the antibody has been validated for your specific application (WB, ELISA, IHC, flow cytometry)

• Clonality considerations:

  • Monoclonal antibodies: Better for specific epitope detection and reproducibility

  • Polyclonal antibodies: Advantageous for detecting denatured proteins or low-abundance targets

• Epitope location: N-terminal vs. C-terminal targeting may yield different results depending on protein processing

• Paired antibody requirements: For sandwich ELISA, select validated antibody pairs with proven compatibility

Methodologically, preliminary validation experiments comparing multiple antibodies on your specific samples are strongly recommended before proceeding to full-scale experiments.

What protocol optimizations are critical for Western blotting with Metrnl antibodies?

Western blotting with Metrnl antibodies requires specific considerations:

• Sample preparation:

  • For secreted Metrnl: Concentrate culture medium using TCA precipitation or commercial concentrators

  • For cellular Metrnl: Standard lysis buffers with protease inhibitors are sufficient

• Gel separation:

  • 10-12% polyacrylamide gels typically provide optimal resolution

  • Extended running times may improve separation of glycosylated forms

• Transfer conditions:

  • Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour

  • PVDF membranes may provide better results than nitrocellulose for Metrnl detection

• Blocking and antibody incubation:

  • 5% non-fat milk in TBST is typically sufficient

  • Primary antibody dilutions ranging from 1:500-1:5000 depending on specific antibody

  • Overnight incubation at 4°C often improves sensitivity

• Molecular weight interpretation:

  • Expect bands between 34-43 kDa for glycosylated forms

  • Multiple bands may represent different post-translational modifications rather than non-specific binding

How can researchers optimize ELISA protocols using Metrnl antibodies?

ELISA optimization requires careful consideration of antibody pairs and detection systems:

• Sandwich ELISA design:

  • Use validated antibody pairs (capture/detection) targeting different epitopes

  • Example: MAB7867 as capture antibody paired with MAB78671 as detection antibody

• Standard curve preparation:

  • Recombinant Metrnl protein should be serially diluted 2-fold

  • Include concentration range spanning expected physiological levels

• Protocol optimization:

  • Coating concentration: Typically 1-4 μg/mL of capture antibody

  • Sample dilution: Optimize based on expected Metrnl concentration

  • Detection antibody: Biotinylation often improves sensitivity

  • Signal development: Streptavidin-HRP followed by appropriate substrate

• Sensitivity considerations:

  • Detection limits should be experimentally determined

  • Consider sample concentration techniques for low-abundance samples

How can Metrnl antibodies be utilized to investigate adipose tissue biology?

Metrnl antibodies have proven valuable in exploring adipose tissue biology through several methodological approaches:

• Immunohistochemical analysis:

  • Allows visualization of Metrnl distribution throughout adipose tissue

  • Studies show Metrnl is distributed throughout adipose tissue (excluding lipid droplets)

  • Enables comparison between white and brown adipose depots

• Functional blocking studies:

  • Anti-Metrnl antibodies can block protein function, revealing physiological roles

  • Blocking antibodies have demonstrated inhibition of adipose thermogenesis during cold exposure

  • Can inhibit M2 macrophage activation induced by Metrnl

• Expression analysis:

  • Western blotting of adipose tissue fractions (adipocytes vs. stromal cells)

  • Studies show comparable expression between adipocytes and stromal cells

  • Lower expression in unactivated macrophages compared to adipocytes

• Secretome analysis:

  • Detection of secreted Metrnl in adipose tissue explant medium

  • Metrnl protein is detectably secreted from white adipose tissue in culture

What are the methodological approaches for studying Metrnl's role in immune modulation?

Research into Metrnl's immunomodulatory functions employs several antibody-dependent techniques:

• Immune cell phenotyping:

  • Flow cytometry with Metrnl antibodies for detection in immune cell populations

  • Antibodies enable characterization of Metrnl-expressing cells during inflammation

• Cytokine response assessment:

  • Blocking antibodies reveal Metrnl's impact on cytokine production

  • Studies show anti-Metrnl antibodies reduce IL-4/IL-13 expression and eosinophil recruitment

  • Affects M2 macrophage polarization markers

• In vivo blocking studies:

  • Administration of anti-Metrnl antibodies in animal models

  • Has revealed Metrnl's role in cold adaptation

  • Demonstrated involvement in Type 2 immune responses

• Autoimmune disease models:

  • Detecting altered Metrnl expression in autoimmune conditions

  • Antibodies reveal Metrnl's potential role in Type 1 diabetes, thyroid autoimmune diseases, and rheumatoid arthritis

  • Enables correlation with disease markers and progression

How can researchers investigate Metrnl's post-translational modifications using antibodies?

Investigating Metrnl's post-translational modifications requires specialized approaches:

• Glycosylation analysis:

  • Western blotting with Metrnl antibodies reveals multiple bands (34-43 kDa)

  • Enzymatic deglycosylation followed by immunoblotting can identify glycosylation sites

  • Comparison of predicted vs. observed molecular weights provides insights into modification extent

• Secretion pathway studies:

  • Intracellular vs. extracellular immunostaining distinguishes maturation stages

  • Antibodies specific to different regions may differentially detect processed forms

• Mass spectrometry validation:

  • Immunoprecipitation with Metrnl antibodies followed by mass spectrometry

  • Multiple bands recognized by anti-Metrnl antibodies have been confirmed as Metrnl by mass spectrometry

Why might researchers observe multiple bands when using Metrnl antibodies in Western blotting?

Multiple bands in Metrnl Western blots may reflect biological reality rather than technical issues:

• Post-translational modifications:

  • Glycosylation produces bands between 34-43 kDa despite calculated weight of ~30 kDa

  • Two distinct bands between 34-43 kDa have been confirmed as Metrnl by mass spectrometry

• Proteolytic processing:

  • Signal peptide cleavage (45 amino acids) produces mature 266 amino acid protein

  • Potential additional processing during secretion or tissue-specific modification

• Technical considerations:

  • Sample preparation method may affect observed pattern

  • Reducing vs. non-reducing conditions might reveal different forms

  • Gel percentage and running conditions can affect band resolution

• Validation approaches:

  • Compare multiple antibodies targeting different epitopes

  • Include recombinant protein control

  • Consider immunoprecipitation followed by mass spectrometry for band identification

What controls are essential when using Metrnl antibodies in various applications?

Robust experimental design requires appropriate controls:

• Western blotting controls:

  • Positive control: Recombinant Metrnl protein or tissue with confirmed expression

  • Negative control: Tissue from Metrnl knockout models when available

  • Primary antibody omission control

  • Blocking peptide competition control when available

• ELISA controls:

  • Standard curve using recombinant protein

  • Blank wells (no sample)

  • Known positive and negative samples

  • Inter-assay control samples for plate-to-plate comparison

• Immunohistochemistry controls:

  • Isotype control antibody

  • Primary antibody omission

  • Tissue from Metrnl knockout models when available

  • Positive tissue controls with established expression patterns

• Flow cytometry controls:

  • Isotype-matched control antibody

  • Unstained cells

  • Single-color controls for compensation

  • Fixed/permeabilized cells for intracellular staining

How should researchers interpret contradictory results when using different Metrnl antibodies?

Contradictory results with different antibodies require systematic investigation:

• Epitope mapping consideration:

  • Antibodies targeting different epitopes may yield different results

  • N-terminal vs. C-terminal targeting can affect detection of processed forms

  • Some epitopes may be masked by protein-protein interactions or conformational changes

• Methodological reconciliation approach:

  • Compare antibody validation data and target epitope information

  • Evaluate species reactivity claims vs. actual experimental system

  • Consider antibody format (polyclonal vs. monoclonal) and host species

• Complementary technique validation:

  • Supplement antibody-based detection with mRNA expression analysis

  • Consider alternative detection methods (mass spectrometry)

  • Use genetic approaches (knockout/knockdown) to validate antibody specificity

• Literature reconciliation:

  • Compare with published findings using the same antibodies

  • Consider tissue-specific or context-dependent expression patterns

  • Evaluate experimental conditions that might affect Metrnl expression or detection

How can Metrnl antibodies contribute to understanding the protein's role in disease pathogenesis?

Metrnl antibodies enable mechanistic investigation in multiple disease contexts:

• Autoimmune disease investigation:

  • Metrnl shows altered expression in several autoimmune conditions

  • Type 1 diabetes: Metrnl treatment delays onset and reduces islet lymphocyte infiltration

  • Thyroid autoimmunity: Decreased Metrnl levels observed in Graves' disease

  • Rheumatoid arthritis: Increased expression in synovial membranes

• Inflammatory skin disorders:

  • Upregulated Metrnl expression observed in:

    • Familial primary localized cutaneous amyloidosis

    • Psoriasis

    • Prurigo nodularis

    • Actinic keratosis

    • Atopic dermatitis

• Cancer research applications:

  • Increased immunoreactivity in:

    • Invasive ductal breast cancer

    • Basal cell carcinoma

    • Trichoblastoma

    • Malignant mesothelioma

    • Serous ovarian tumors

  • Potential prognostic marker in bladder cancer

• Mechanistic studies using blocking antibodies:

  • Anti-Metrnl antibodies reveal functional roles in:

    • Type 2 immune responses

    • Macrophage polarization

    • Inflammatory cytokine production

What are the latest methodological approaches for studying Metrnl using antibody-based techniques?

Cutting-edge approaches leverage Metrnl antibodies in novel ways:

• Single-cell analysis:

  • Flow cytometry with Metrnl antibodies identifies specific expressing cell populations

  • Intracellular staining protocols for Metrnl enable phenotypic correlation

  • Cell sorting based on Metrnl expression allows transcriptomic profiling

• In vivo imaging:

  • Fluorescently labeled Metrnl antibodies for dynamic imaging

  • Tracking Metrnl expression changes during physiological challenges

  • Correlating cellular distribution with functional responses

• Therapeutic targeting strategies:

  • Blocking antibodies demonstrate potential intervention points

  • Anti-Metrnl antibodies affect thermogenesis and immune responses

  • Potential applications in inflammatory and metabolic conditions

• Biomarker development:

  • ELISA development for clinical sample analysis

  • Correlation of circulating Metrnl levels with disease states

  • Identification of Metrnl as potential therapeutic target or diagnostic indicator

How can researchers use antibodies to study the relationship between Metrnl and other signaling pathways?

Investigating Metrnl's relationship with other pathways involves sophisticated antibody applications:

• Signaling pathway analysis:

  • Metrnl has been linked to AMPK-PAK2 pathway in cardiomyocyte protection

  • Associated with AMPK-mTOR-p70S6K pathway in intestinal inflammation

  • Connected to Stat3/IGF-1 signaling in muscle regeneration

  • Involved in AMPK and PPARδ pathways in endothelial inflammation

• Co-immunoprecipitation approaches:

  • Identifying Metrnl-interacting proteins using antibody-based pull-down

  • Revealing protein complexes formed during signaling events

  • Validating direct protein-protein interactions

• Phospho-signaling correlation:

  • Combining Metrnl antibodies with phospho-specific antibodies

  • Mapping temporal relationships between Metrnl expression and pathway activation

  • Establishing causality through blocking antibody studies

• Receptor identification strategies:

  • Receptor-ligand interaction studies using tagged Metrnl and antibody detection

  • Cross-linking approaches followed by immunoprecipitation

  • Competition assays with blocking antibodies to identify functional binding partners