GAS6 Antibody

What is GAS6 protein and why is it a significant research target?

GAS6 (Growth Arrest Specific 6) is a 75 kDa multimodular protein upregulated by various cell types in response to growth arrest. It functions as a ligand for tyrosine-protein kinase receptors AXL, TYRO3, and MER, whose signaling pathways regulate cell growth, survival, adhesion, and migration . GAS6 has a complex structure featuring:

• A gamma-carboxylated N-terminal Gla domain

• Four EGF-like repeats

• A C-terminal region with homology to steroid hormone binding globulin (SHBG)

The GAS6/AXL signaling axis plays critical roles in:

• Endothelial cell survival during acidification

• Cytokine signaling during natural killer cell development

• Hepatic regeneration

• Neuron survival and migration

• Platelet activation and thrombotic response regulation

Its involvement in these diverse physiological processes and in pathological conditions like cancer and inflammation makes GAS6 a valuable research target.

How should researchers select the appropriate GAS6 antibody for specific experimental applications?

Selection of the optimal GAS6 antibody requires consideration of multiple factors:

For studies focusing on specific GAS6 functions, consider antibodies targeting the LG domains, as they mediate receptor binding. The CNTO300 antibody, for example, recognizes a peptide sequence in the LG1 domain that affects receptor binding .

How can researchers validate the specificity of their GAS6 antibodies?

Methodological approach to GAS6 antibody validation:

• Positive and negative controls:

  • Use cell lines with known GAS6 expression (e.g., SH-SY5Y, SW480, COLO 205)

  • Include knockout or knockdown samples (e.g., siGAS6 transfected cells)

• Multiple detection methods:

  • Perform Western blotting to confirm molecular weight (expected bands at ~75 kDa, with possible variants at ~64 kDa and ~50 kDa)

  • Conduct immunoprecipitation followed by mass spectrometry

• Cross-reactivity assessment:

  • Test against recombinant human and mouse GAS6 proteins (some antibodies show ~30% cross-reactivity)

  • Evaluate potential cross-reactivity with Protein S (43% amino acid identity with human GAS6)

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide or recombinant GAS6 before application

  • Observe signal reduction to confirm specificity

What are the optimal conditions for detecting GAS6 using Western blotting?

Optimized Western blot protocol for GAS6 detection:

• Sample preparation:

  • For cell lysates: Use RIPA buffer with protease inhibitors

  • For secreted GAS6: Concentrate conditioned media using anti-FLAG affinity gel for FLAG-tagged GAS6

• Electrophoresis conditions:

  • Use 8-10% SDS-PAGE gels to resolve the 75 kDa GAS6 protein

  • Include reducing conditions (β-mercaptoethanol)

• Transfer parameters:

  • Transfer to PVDF membrane (preferred over nitrocellulose)

  • Use wet transfer at 30V overnight at 4°C for optimal transfer of large proteins

• Blocking and antibody incubation:

  • Block with 5% BSA in TBST (avoid milk for phosphorylated proteins)

  • Primary antibody dilutions: 1:500-1:1000 for optimal signal-to-noise ratio

  • Incubate overnight at 4°C for maximum sensitivity

• Detection system:

  • HRP-conjugated secondary antibodies with enhanced chemiluminescence

  • For multiplexing, consider fluorescent secondary antibodies

Expected results: GAS6 typically appears as a band at ~75 kDa, with possible additional bands at ~64 kDa and ~50 kDa representing alternative splicing variants or proteolytic fragments .

How should researchers design experimental controls when studying GAS6/AXL signaling in cancer models?

Comprehensive control strategy for GAS6/AXL signaling experiments:

Using this control framework enables proper attribution of observed effects to GAS6/AXL signaling rather than non-specific or alternative pathways.

What is the recommended protocol for GAS6 immunohistochemistry in tissue samples?

Optimized IHC protocol for GAS6 detection in tissue sections:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Embed in paraffin and section at 4-5 μm thickness

• Antigen retrieval (critical step):

  • Heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (pH 9.0)

  • Alternative: citrate buffer (pH 6.0) for certain antibodies

  • Heat at 95-98°C for 20 minutes

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂

  • Block non-specific binding with 5% normal serum

  • Primary antibody dilution: 1:50-1:500 (optimize for each antibody)

  • Incubate overnight at 4°C for maximum sensitivity and specificity

• Detection system:

  • HRP-DAB Cell & Tissue Staining Kit for chromogenic detection

  • Counterstain with hematoxylin for nuclear visualization

• Controls and interpretation:

  • Positive control: Human stomach cancer tissue (known to express GAS6)

  • Negative control: Primary antibody omission

  • Interpretation: Evaluate intensity and distribution of staining (membranous, cytoplasmic, nuclear)

Expected staining patterns: GAS6 typically shows cytoplasmic and/or extracellular matrix staining in positive tissues. In tumor specimens, evaluate both tumor cells and stromal compartments, as GAS6 expression in the stroma has been associated with AXL expression in tumor cells .

How can researchers investigate the functional interaction between GAS6 and its receptors?

Methodological approaches to study GAS6-receptor interactions:

• Binding kinetics analysis:

  • Surface Plasmon Resonance (BIAcore):

    • Immobilize receptor-Fc fusion proteins (Axl-Fc, Dtk-Fc, Mer-Fc) on sensor chips

    • Measure binding of recombinant GAS6 at different concentrations

    • Determine association/dissociation constants

• Domain-specific binding studies:

  • Express individual GAS6 domains (LG1, LG2) and test receptor binding

  • Use domain-specific antibodies to block binding and identify critical regions

  • Create chimeric proteins to map interaction interfaces

• Co-immunoprecipitation approaches:

  • Precipitate receptor complexes from cells expressing GAS6 and receptors

  • Confirm interaction by Western blotting

  • Identify additional complex components by mass spectrometry

• Functional consequence assessment:

  • Compare receptor phosphorylation induced by full-length GAS6 versus domain fragments

  • Measure downstream signaling activation (AKT, ERK pathways)

  • Quantify biological responses (survival, migration, phagocytosis)

Studies have revealed that contrary to previous assumptions, the LG1 domain contains a second receptor-binding site distinct from the previously identified site in the LG2 domain , highlighting the importance of comprehensive domain analysis.

What techniques should be employed to investigate GAS6's role in the tumor microenvironment?

Multi-disciplinary approach to studying GAS6 in the tumor microenvironment:

• Spatial expression analysis:

  • Multiplex immunohistochemistry for simultaneous detection of:

    • GAS6 (stromal vs. tumor cells)

    • TAM receptors (AXL, TYRO3, MER)

    • Immune cell markers (macrophages, NK cells, T cells)

  • In situ hybridization to detect GAS6 mRNA in specific cell populations

  • Laser capture microdissection for region-specific expression analysis

• Secretome analysis:

  • Measure GAS6 in conditioned media from tumor and stromal cells

  • Examine cytokine profiles affected by GAS6/AXL signaling (IL-4, CCL3-5, G-CSF)

  • Analyze exosomal GAS6 and soluble receptors as decoy mechanisms

• Immune modulation assessment:

  • Flow cytometry to quantify immune cell populations in presence/absence of GAS6

  • Functional assays to measure:

    • T cell activation

    • Macrophage polarization

    • NK cell cytotoxicity

  • Analysis of MHC-I and PD-L1 expression on tumor cells

• In vivo models:

  • GAS6 knockout mice or conditional knockout models

  • Neutralizing antibody treatment in tumor-bearing mice

  • Analysis of tumor growth, metastasis, and immune infiltration

Research has demonstrated that the GAS6/AXL pathway promotes an immunosuppressive tumor microenvironment by regulating the expression of immune checkpoint molecules and anti-inflammatory cytokines .

How do researchers troubleshoot inconsistent GAS6 antibody performance in experimental applications?

Systematic troubleshooting approach for GAS6 antibody applications:

• Western blot issues and solutions:

• Immunohistochemistry optimization:

• Immunoprecipitation considerations:

  • Use agarose-conjugated antibodies for cleaner pull-downs

  • Pre-clear lysates to reduce non-specific binding

  • Consider native conditions to preserve protein-protein interactions

• Cross-reactivity management:

  • Test multiple antibodies targeting different epitopes

  • Perform peptide competition assays

  • Validate with knockout/knockdown controls

What considerations are important when developing a GAS6 ELISA assay for clinical research?

Critical parameters for developing robust GAS6 ELISA assays:

• Antibody pair selection:

  • Capture antibody: Use antibodies targeting stable epitopes unaffected by sample processing

  • Detection antibody: Select antibodies recognizing distinct epitopes from capture antibody

  • Validated pairs: Consider using established combinations (e.g., MAB8851/8852/8853 with AF885)

• Sample preparation optimization:

  • Plasma: Use citrated plasma rather than serum to avoid platelet activation

  • Tissue lysates: Standardize extraction buffer and protein concentration

  • Spike-and-recovery experiments to validate matrix effects

• Assay standardization:

  • Recombinant GAS6 standard curve (range: 31.2-2,000 pg/mL)

  • Include internal quality controls at low, medium, and high concentrations

  • Establish minimum required dilution for each sample type

• Technical validation parameters:

  • Precision: Intra-assay CV <10%, inter-assay CV <15%

  • Accuracy: Recovery 80-120% of spiked standards

  • Sensitivity: Determine LLOQ based on standard curve precision profile

  • Specificity: Verify lack of interference from related proteins (Protein S)

• Clinical considerations:

  • Pre-analytical variables: Standardize sample collection and processing

  • Reference range establishment: Analyze samples from healthy individuals

  • Disease state evaluation: Compare GAS6 levels in normal vs. pathological conditions

For clinical research applications, consider commercial ELISA development kits like the Human Gas6 DuoSet ELISA Kit (DY885B) that provide validated reagents and protocols .

How can GAS6 antibodies be utilized to investigate viral entry mechanisms?

Methodological framework for studying GAS6-mediated viral entry:

• Binding studies:

  • Assess GAS6 binding to viral phosphatidylserine using:

    • Solid-phase binding assays with purified phosphatidylserine

    • Co-immunoprecipitation of GAS6 with viral particles

  • Block binding with GAS6 neutralizing antibodies to confirm specificity

• Receptor bridging analysis:

  • Investigate GAS6's ability to bridge viral phosphatidylserine to TAM receptors

  • Use domain-specific antibodies to identify regions involved in viral binding

  • Compare with Protein S bridging to understand specificity

• Viral entry assays:

  • Quantify viral infection in presence/absence of GAS6

  • Add GAS6 neutralizing antibodies to block entry

  • Use receptor-specific inhibitors to determine relative importance of AXL vs. other TAM receptors

• Mechanistic studies:

  • Examine the role of GAS6 gamma-carboxylation in viral binding

  • Investigate downstream signaling pathways activated during viral entry

  • Compare entry mechanisms across different virus families

Research has demonstrated that GAS6 facilitates viral entry through "apoptotic mimicry" for multiple viruses, including dengue virus, vaccinia virus, ebola virus, and marburg virus . This mechanism represents a potential therapeutic target for antiviral development.

What strategies should be employed to study GAS6 isoforms and post-translational modifications?

Comprehensive approach to analyzing GAS6 variants and modifications:

• Isoform identification:

  • RT-PCR with isoform-specific primers to detect splice variants

  • Western blotting with domain-specific antibodies to identify protein isoforms

  • Mass spectrometry for detailed isoform characterization

• Gamma-carboxylation analysis:

  • Comparison of GAS6 from vitamin K-treated vs. untreated cells

  • Conformation-specific antibodies to detect properly carboxylated GAS6

  • Functional assays to assess carboxylation-dependent activities

• Proteolytic processing detection:

  • N-terminal and C-terminal specific antibodies to identify fragments

  • Analysis of SHBG domain release and its independent activity

  • In vitro proteolysis with different proteases to map cleavage sites

• Functional consequences:

  • Compare receptor binding affinities of different isoforms

  • Assess calcium-dependence of receptor interactions

  • Evaluate biological activities in cellular assays

Studies have shown that GAS6 displays different receptor-binding properties depending on its post-translational modifications, with gamma-carboxylation of the Gla domain being critical for several biological functions including phagocytosis and VEGF-induced endothelial chemotaxis inhibition .

How can researchers investigate the interplay between GAS6/AXL signaling and immune checkpoint pathways?

Integrated methodological approach to study GAS6/AXL and immune checkpoint interactions:

• Expression correlation analysis:

  • Multiplex immunohistochemistry for co-localization of:

    • GAS6 and TAM receptors

    • PD-1/PD-L1 and other immune checkpoints

    • Infiltrating immune cell markers

  • Single-cell RNA sequencing to identify co-expression patterns

• Signaling pathway interconnections:

  • Phosphoproteomic analysis of shared downstream mediators

  • Inhibitor studies (AXL inhibitors ± immune checkpoint inhibitors)

  • Time-course experiments to determine signaling sequence

• Functional immune assays:

  • T cell activation and proliferation in presence of GAS6

  • Measure PD-L1 expression changes after GAS6 stimulation

  • Assessment of antigen presentation via MHC-I expression analysis

• Therapeutic combination strategies:

  • Synergy studies with AXL inhibitors and immune checkpoint blockers

  • In vivo tumor models to assess combinatorial treatment efficacy

  • Analysis of immune infiltrate changes with combined targeted therapy

Research has established that GAS6/AXL signaling regulates the expression of immune checkpoint molecules like PD-L1 and affects MHC-I presentation, suggesting that targeting this pathway could enhance the efficacy of immunotherapies .