MERTK Monoclonal Antibody

What is MERTK and why is it significant as a research target?

MERTK is a transmembrane receptor tyrosine kinase belonging to the TAM (Tyro3, Axl, MerTK) family. It plays crucial roles in multiple physiological processes including:

• Clearance of apoptotic cells (efferocytosis)

• Regulation of immune tolerance and inflammatory responses

• Cell survival, migration, and differentiation

• Phagocytosis pathways, particularly in the retinal pigment epithelium

MERTK's significance stems from its involvement in various diseases when dysregulated, including cancer, autoimmune disorders, and inflammatory conditions. The receptor transduces signals from the extracellular matrix by binding to ligands such as Gas6, Protein S, LGALS3, TUB, and TULP1 .

What are the key applications for MERTK monoclonal antibodies in experimental research?

MERTK monoclonal antibodies have been validated for numerous research applications:

Different antibody clones may perform optimally in specific applications, so researchers should select antibodies validated for their intended use .

How do I determine the optimal antibody dilution for my experimental system?

Determining optimal antibody dilution requires systematic titration:

• Begin with the manufacturer's recommended dilution range (e.g., 1:500-1:1000 for Western blotting)

• Perform a dilution series (e.g., 1:250, 1:500, 1:1000, 1:2000)

• Include appropriate positive controls (e.g., Hep G2 cells for human MERTK antibodies)

• Include negative controls (cells without MERTK expression or MERTK knockout samples)

• Select the dilution that provides optimal signal-to-noise ratio

Remember that optimal dilutions may vary between different batches of antibodies and different experimental systems. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test for flow cytometry applications .

How do I confirm the specificity of MERTK antibodies in my experimental system?

Confirming antibody specificity is critical for reliable results:

• Use positive and negative cell lines or tissues (e.g., MERTK is highly expressed in macrophages but not in dendritic cells)

• Perform knockdown or knockout validation (using MERTK siRNA or CRISPR-edited cells)

• Verify binding to recombinant MERTK protein of the appropriate species

• Test cross-reactivity with other TAM family members (Axl and Tyro3)

• Use multiple antibody clones targeting different epitopes

Studies have shown that high-quality MERTK antibodies selectively bind to MERTK but not to other family members like AXL and TYRO3. This selectivity can be confirmed using ELISA against recombinant proteins .

What explains the discrepancy between calculated and observed molecular weights for MERTK in Western blot analysis?

The calculated molecular weight of MERTK is approximately 110 kDa, but researchers often observe bands at 150-200 kDa in Western blot analysis . This discrepancy is due to:

• Post-translational modifications, primarily extensive glycosylation

• Potential dimerization or complex formation

• Splice variants of MERTK

• Cell type-specific modifications

To address this discrepancy:

• Include appropriate positive controls with known MERTK expression

• Consider deglycosylation experiments to confirm identity

• Use multiple antibodies targeting different MERTK epitopes

• Perform peptide competition assays to confirm specificity

What are the key considerations for immunohistochemistry applications with MERTK antibodies?

When performing immunohistochemistry with MERTK antibodies:

• Fixation conditions: Some MERTK antibodies work better with specific fixation methods

  • The DS5MMER antibody clone works on paraformaldehyde-fixed cells

  • Some antibodies may require antigen retrieval techniques

• Cross-reactivity: Verify species reactivity

  • Certain antibodies (like clone 20F5) cross-react with both human and mouse MERTK

  • Others are species-specific (human-only or mouse-only)

• Control tissues:

  • Use tissues with known MERTK expression (e.g., macrophage-rich tissues)

  • Include MERTK-knockout tissues when available

• Signal amplification:

  • May be necessary for detecting low expression levels

  • Consider tyramide signal amplification for enhanced sensitivity

How do cellular localization patterns affect MERTK antibody selection and experimental design?

MERTK exhibits specific cellular localization patterns that impact experimental design:

• Membrane localization: MERTK is primarily a single-pass type I membrane protein

  • Flow cytometry: Use non-permeabilized cells to detect surface expression

  • Confocal microscopy: Co-stain with membrane markers

• Constitutive shedding: MERTK is constitutively released from the cell surface by metalloproteinases

  • Check culture medium/serum for soluble MERTK

  • LPS stimulation enhances this release

  • Consider this when interpreting flow cytometry data

• Intracellular signaling: Following activation, MERTK undergoes internalization

  • May require permeabilization for detection of internalized receptor

  • Time-course experiments can track receptor internalization

Understanding these localization patterns is essential for proper experimental design and interpretation of results .

How can MERTK agonistic antibodies be used for targeted therapeutic development?

MERTK agonistic antibodies represent a novel approach for targeted therapeutics:

• Mechanism of action:

  • Agonistic antibodies activate MERTK signaling without requiring natural ligands

  • This activation can be measured by increased phospho-AKT (Ser473) in macrophages

  • Unlike Fcγ receptor-mediated phagocytosis, MERTK-mediated clearance is immunologically silent

• Bispecific antibody approach:

  • Bispecific antibodies can be engineered using knob-hole technology

  • One arm targets MERTK (agonistic function)

  • The other arm targets a disease-specific antigen (e.g., CD20 on B cells or amyloid beta)

  • This approach enables targeted clearance without inducing proinflammatory cytokine release

• Screening methodology:

  • Cell-based assays measuring phospho-AKT increase in macrophages

  • Validation using MERTK small molecule inhibitors to confirm specificity

  • Surface plasmon resonance to measure binding kinetics (kon, koff, KD)

This approach has shown promise for targeted removal of protein aggregates in neurodegenerative diseases and for targeting cancer cells while avoiding inflammatory side effects .

What role does MERTK play in cancer progression and how can antibodies be used to study this process?

MERTK's involvement in cancer progression can be studied using monoclonal antibodies:

• Aberrant expression in malignancies:

  • MERTK is overexpressed in various solid and hematological cancers

  • Associated with reduced apoptosis, increased metastasis, and drug resistance

• Signaling pathways:

  • MERTK activates PI3K/AKT and MAPK pathways in cancer cells

  • Promotes cell survival, proliferation, and migration

  • Antibodies can be used to block these signaling pathways

• Experimental approaches:

  • Knockdown studies: Using anti-MERTK to confirm specificity of MERTK-targeted shRNA

  • Phosphorylation analysis: Monitoring activation status after drug treatment

  • Functional assays: Migration, invasion, apoptosis resistance

  • Tumor microenvironment studies: MERTK's role in immune evasion

• Therapeutic implications:

  • ADC development: RGX-019-MMAE (an antibody-drug conjugate) shows promise in AML

  • Combination therapies: Anti-MERTK with chemotherapy in B-ALL enhances sensitivity

Studies show that targeting MERTK in AML cell lines led to increased myeloblast apoptosis and decreased colony formation, while mice transplanted with MERTK-knockdown AML cells showed prolonged survival .

How can MERTK antibodies be optimized for flow cytometry and sorting of tissue-resident macrophages?

Optimizing MERTK antibodies for flow cytometry of tissue-resident macrophages:

• Panel design considerations:

  • MERTK can help discriminate macrophages from dendritic cells

  • Combine with other macrophage markers (F4/80, CD11b, CD64)

  • Consider fluorophore selection based on instrument configuration:

    • APC (Excitation: 633-647 nm; Emission: 660 nm)

    • PE (Blue Laser: 488 nm; Green Laser: 532 nm)

    • Alexa Fluor 700 (Emission: 723 nm)

• Sample preparation:

  • Paraformaldehyde fixation is compatible with DS5MMER clone

  • Constitutive shedding of MERTK may affect detection in cultured cells

  • Fresh samples typically yield better results than frozen

• Titration strategy:

  • For resident peritoneal macrophages: ≤0.5 μg/test

  • Define test as antibody amount to stain cells in 100 μL final volume

  • Cell numbers from 10^5 to 10^8 cells/test

• Controls:

  • FMO (Fluorescence Minus One) controls

  • MERTK-deficient cells to establish background

  • Blocking experiments to confirm specificity

What epitope considerations are important when selecting MERTK antibodies for functional studies?

Epitope considerations critically impact the functional outcomes of MERTK antibodies:

• Epitope mapping techniques:

  • Microarray-based microfluidic systems (e.g., IBIS-MX96 SPRi)

  • Epitope binning through competitive binding assays

  • Surface plasmon resonance to determine binding kinetics

• Functional domains:

  • Extracellular domain: Contains two immunoglobulin-like C2-type domains and two fibronectin type-III domains

  • Kinase domain: Critical for signaling function

  • Antibodies targeting different domains have distinct functional effects

• Agonistic vs. antagonistic activity:

  • Agonistic antibodies (e.g., clones 18G7, 14C3, 20F5) induce MerTK phosphorylation

  • Antagonistic antibodies (e.g., DS5MMER) can inhibit Gas6-induced MERTK phosphorylation

  • Some antibodies (like DS5MMER) have IC50 < 5 nM for inhibiting Gas6-induced phosphorylation

• Cross-reactivity between species:

  • Some antibodies (like clone 20F5) cross-react with both human and mouse MERTK

  • Others are species-specific, limiting translational studies

  • Sequence alignment and structural analysis can predict cross-reactivity

Understanding these epitope considerations enables selection of antibodies with appropriate functional characteristics for specific research questions.

How can MERTK monoclonal antibodies be used to study the role of MERTK in T cell tolerance?

MERTK's role in T cell tolerance can be investigated using monoclonal antibodies:

• Experimental design for studying AC-induced T cell tolerance:

  • MERTK mediates apoptotic cell (AC)-induced inhibition of dendritic cell (DC) activation

  • This process is critical for preventing autoimmunity

  • Key readouts include:

    • Proinflammatory cytokine secretion

    • Costimulatory molecule expression (flow cytometry)

    • T cell activation (proliferation assays)

• Methodological approach:

  • Compare wild-type vs. MERTK-deficient DCs

  • Use anti-MERTK antibodies to block interaction with ACs

  • Analyze Gas6 (MERTK ligand) involvement using neutralizing antibodies

  • Track phosphatidylserine recognition using fluorescent annexin V

• Disease models:

  • NOD (non-obese diabetic) mice show exacerbated autoimmune diabetes in absence of MERTK

  • Adoptive transfer of β cell-specific CD4+ T cells into MERTK-deficient mice

  • Analysis of pancreatic DC activation status using flow cytometry with anti-MERTK

Studies have demonstrated that MERTK-deficient DCs show resistance to AC-induced inhibition, leading to increased T cell activation and autoimmune pathology .

What are the considerations for developing MERTK antibody-drug conjugates (ADCs) for cancer therapy?

Development of MERTK antibody-drug conjugates requires several considerations:

• Antibody selection criteria:

  • High affinity and specificity for MERTK

  • Internalization efficiency (critical for ADC functionality)

  • Cross-reactivity with mouse MERTK (for preclinical studies)

  • Epitope selection to maximize tumor targeting

• Conjugation strategy:

  • Payload selection (e.g., MMAE in RGX-019-MMAE)

  • Linker chemistry (cleavable vs. non-cleavable)

  • Drug-to-antibody ratio optimization

  • Site-specific conjugation vs. random conjugation

• Preclinical testing methodology:

  • In vitro cytotoxicity in MERTK-expressing cell lines

  • Comparison with isotype control ADCs

  • Time-course experiments (72h and 120h treatment)

  • Primary patient samples testing

• Tumor targeting specificity:

  • MERTK expression in various AML subsets

  • Reverse Phase Protein Arrays (RPPA) to analyze protein expression

  • Flow cytometry to measure MERTK expression in leukemic cell lines and patient samples

Studies with RGX-019-MMAE have shown promising results in AML, demonstrating the potential of MERTK as a target for ADC development .

How are MERTK recombinant monoclonal antibodies produced and characterized for research applications?

The production and characterization of MERTK recombinant monoclonal antibodies involves:

• Production process:

  • Gene sequencing of the MERTK monoclonal antibody

  • Cloning into plasmid vectors

  • Transfection into host cell lines

  • Purification from cell culture supernatant via affinity chromatography

  • Final testing and characterization

• Characterization methods:

  • SDS-PAGE for purity assessment (>90% purity standard)

  • HPLC for aggregation analysis (<10% aggregation)

  • 0.2 μm post-manufacturing filtration

  • Binding kinetics determination using surface plasmon resonance

  • Epitope binning using microarray-based methods

• Quality control criteria:

  • Reactivity testing against target species

  • Cross-reactivity testing with other TAM family members

  • Functional validation in relevant applications

  • Lot-to-lot consistency assessment

  • Stability testing under various storage conditions

• Application-specific validation:

  • Western blot: Confirming expected molecular weight (calculated vs. observed)

  • Flow cytometry: Testing on cells with known MERTK expression

  • Immunoprecipitation: Verifying pull-down efficiency

  • ELISA: Determining sensitivity and specificity

This rigorous production and characterization process ensures antibodies of consistent quality for research applications.

What are common challenges in Western blotting with MERTK antibodies and how can they be addressed?

Common challenges and solutions for MERTK Western blotting:

• Multiple bands or unexpected molecular weight:

  • MERTK's calculated MW is 110 kDa, but observed MW is 150-200 kDa due to glycosylation

  • Solution: Include positive control (e.g., Hep G2 cells for human MERTK)

  • Consider deglycosylation treatment to confirm identity

  • Use reducing and non-reducing conditions to assess multimeric forms

• Weak or no signal:

  • MERTK expression can vary significantly between cell types

  • Solution: Enrich for membrane fraction in sample preparation

  • Optimize antibody concentration (1:500-1:1000 dilution recommended)

  • Consider longer exposure times or more sensitive detection methods

  • Verify sample preparation method preserves membrane proteins

• High background:

  • Solution: Increase blocking time/concentration

  • Optimize antibody dilution and washing steps

  • Use highly purified antibody preparations

  • Consider alternative blocking agents (BSA vs. milk)

• Inconsistent results between experiments:

  • MERTK shedding can affect levels in different culture conditions

  • Solution: Standardize culture conditions and sample preparation

  • Consider analyzing both cell lysates and culture supernatants

  • Monitor time between cell collection and lysis to minimize proteolysis

How can researchers optimize immunohistochemistry protocols for detecting MERTK in different tissue contexts?

Optimizing immunohistochemistry protocols for MERTK detection:

• Fixation and sample preparation:

  • Paraformaldehyde fixation works well with many MERTK antibodies

  • For frozen sections: Test antibodies specifically validated for IHC-Fr

  • For paraffin sections: Optimize antigen retrieval methods (heat-induced vs. enzymatic)

  • Section thickness: 5-7 μm sections typically provide optimal results

• Protocol optimization:

  • Blocking endogenous peroxidase activity is critical

  • Antibody concentration: Start with manufacturer recommendations and titrate

  • Incubation time and temperature: Overnight at 4°C often yields best results

  • Signal amplification: Consider tyramide signal amplification for low expression

• Tissue-specific considerations:

  • Brain tissue: MERTK is expressed in microglia

  • Retina: MERTK is highly expressed in retinal pigment epithelium

  • Spleen/lymphoid tissues: MERTK expression in macrophages

  • Tumor tissues: Variable expression requires careful optimization

• Controls:

  • Positive control tissues with known MERTK expression

  • Negative controls (isotype control and MERTK-deficient tissues)

  • Peptide competition to confirm specificity

  • Sequential sections with different antibody clones

These optimization strategies ensure reliable and reproducible MERTK detection across different tissue contexts.