GAS6 Antibody, FITC conjugated

What is GAS6 and why is it important in biological research?

GAS6 (Growth Arrest-Specific protein 6) is a member of the vitamin K-dependent protein family found in plasma. It functions as a ligand for TAM (Tyro3, Axl, and Mer) receptors, initiating intracellular tyrosine autophosphorylation and subsequent signal transduction . GAS6 plays critical roles in:

• Modulating innate immunity through TAM signaling

• Maintaining homeostasis of alveolar epithelial cells

• Regulating proliferation and tissue repair processes

• Influencing epithelial-mesenchymal transition (EMT)

Research interest in GAS6 has expanded as it demonstrates protective effects in multiple disease models, including acute lung injury and multi-organ failure syndrome . The availability of FITC-conjugated antibodies has enhanced our ability to visualize and track GAS6 in biological samples.

What are the structural characteristics of GAS6 antibodies used in research?

GAS6 antibodies used in research typically target specific amino acid sequences within the human GAS6 protein. The FITC-conjugated antibody referenced in the search results targets amino acids 70-217 of the human GAS6 protein . Key structural features include:

• Host organism: Commonly rabbit-derived (polyclonal)

• Immunogen: Recombinant human GAS6 protein fragments (typically AA 70-217)

• Conjugation: Fluorescein isothiocyanate (FITC) for direct visualization in fluorescence-based applications

• Purification method: Protein G purification with >95% purity

• Form: Typically provided in liquid form with glycerol-containing buffer

The antibody is designed to recognize human GAS6 with high specificity, making it suitable for various immunological detection methods.

How should GAS6 antibodies be stored to maintain optimal activity?

Proper storage is critical for maintaining antibody functionality. For FITC-conjugated GAS6 antibodies:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles as they can degrade the antibody and diminish the FITC signal

• When stored in the recommended buffer (typically containing 50% glycerol, 0.01M PBS, pH 7.4, and preservatives like 0.03% Proclin 300), the antibody maintains stability for the duration specified by the manufacturer

• For working solutions, store at 4°C protected from light and use within the timeframe recommended by the manufacturer

• Document the date of reconstitution and number of freeze-thaw cycles

Light exposure can diminish FITC fluorescence intensity, so minimize exposure during storage and handling.

How can GAS6 antibody be optimally used for immunofluorescence studies of lung tissue?

When utilizing FITC-conjugated GAS6 antibodies for immunofluorescence studies in lung tissue, particularly in fibrosis models, researchers should consider the following protocol optimizations:

• Tissue preparation:

  • Fix lung tissue sections appropriately (typically 4% paraformaldehyde)

  • Consider antigen retrieval methods if epitope masking is a concern

  • Block with appropriate sera (5-10% normal serum from a species unrelated to the primary antibody)

• Antibody application:

  • Dilution optimization is essential; typically start with 1:100-1:500 and adjust based on signal-to-noise ratio

  • Incubate overnight at 4°C in a humidified chamber protected from light

  • Include appropriate controls (no primary antibody, isotype control)

• Counterstaining considerations:

  • For co-localization studies with epithelial markers, consider combining with anti-E-cadherin (labeled with a non-overlapping fluorophore)

  • For investigating EMT, combine with mesenchymal markers like α-SMA

  • DAPI nuclear counterstain provides cellular context

• Visualization parameters:

  • Use appropriate filter sets for FITC (excitation ~495 nm, emission ~520 nm)

  • Adjust exposure settings to prevent photobleaching

  • Capture multiple fields (≥5) per condition for quantitative analysis

This approach has been successfully employed to demonstrate how GAS6 administration affects EMT markers in bleomycin-induced lung fibrosis models .

What are effective protocols for using GAS6 antibodies in Western blot applications?

Western blot detection of GAS6 requires careful optimization. Based on research applications described in the search results:

• Sample preparation:

  • Extract proteins from cells or tissues using RIPA buffer supplemented with protease and phosphatase inhibitors

  • For detecting GAS6 in cell culture studies, both cell lysates and conditioned media should be analyzed

  • Quantify protein concentration using Bradford or BCA assay

• Gel electrophoresis and transfer:

  • Load 20-50 μg protein per lane

  • Use 8-10% SDS-PAGE gels (GAS6 is ~75-80 kDa)

  • Transfer to PVDF membrane at 100V for 90 minutes

• Immunodetection:

  • Block with 5% non-fat milk or BSA in TBST

  • For detecting GAS6, FITC-conjugated antibodies can be used directly

  • Alternative approach: Use unconjugated primary GAS6 antibody followed by HRP-conjugated secondary antibody

  • Include appropriate positive controls (e.g., recombinant GAS6 or known GAS6-expressing cell lines like LCAFhTERT)

• Signal development and analysis:

  • For chemiluminescence detection, use ECL substrate

  • For direct fluorescence detection with FITC-conjugated antibody, use fluorescence imaging systems

  • Normalize to loading controls such as GAPDH

This protocol has been successfully used to detect GAS6 expression in H1299 NSCLC cells, with LCAFhTERT cells serving as a positive control .

How can the GAS6 antibody be incorporated into flow cytometry assays for apoptosis studies?

FITC-conjugated GAS6 antibodies can be valuable in flow cytometry protocols, particularly when studying GAS6's role in apoptosis:

• Cell preparation:

  • Harvest cells of interest (e.g., primary ATII cells from experimental models)

  • Wash in PBS containing 2% FBS

  • Fix with 2-4% paraformaldehyde if intracellular staining is required

• Staining protocol:

  • For surface staining: Incubate cells with FITC-conjugated GAS6 antibody (1:100-1:200 dilution) for 30-60 minutes at 4°C

  • For intracellular staining: Permeabilize cells with 0.1% saponin or 0.1% Triton X-100 before antibody incubation

  • For dual parameter analysis: Combine with apoptosis markers (e.g., Annexin V-PE and PI)

• Analysis considerations:

  • Set appropriate compensation controls when using multiple fluorophores

  • Establish gating strategy based on forward/side scatter and fluorescence intensity

  • For apoptosis studies, analyze at least 10,000 events per sample

• Experimental design for apoptosis studies:

  • Include positive controls for apoptosis (e.g., staurosporine-treated cells)

  • For studies investigating GAS6's role in protecting against apoptosis, compare GAS6-positive versus GAS6-negative populations

  • Quantify the percentage of cells in early apoptosis (Annexin V+/PI-) and late apoptosis (Annexin V+/PI+)

This approach has been used to demonstrate that recombinant GAS6 administration suppresses apoptosis in primary ATII cells in bleomycin-induced lung fibrosis models, reducing apoptosis from 28.3% to 17.7% .

What factors influence the specificity and sensitivity of FITC-conjugated GAS6 antibodies?

Several factors can impact the performance of FITC-conjugated GAS6 antibodies:

• Antibody characteristics:

  • Polyclonal versus monoclonal nature (polyclonal antibodies offer broader epitope recognition but potentially increased background)

  • The specific immunogen sequence (AA 70-217 for the referenced antibody)

  • Purification method (Protein G purification achieves >95% purity)

  • FITC:protein ratio (optimal conjugation balances fluorescence intensity with antibody function)

• Sample preparation factors:

  • Fixation method and duration (overfixation can mask epitopes)

  • Permeabilization protocol for intracellular targets

  • Blocking effectiveness (insufficient blocking increases non-specific binding)

  • Endogenous biotin or fluorescence in tissues

• Technical variables:

  • Antibody concentration and incubation conditions

  • Buffer composition and pH

  • Washing stringency

  • Exposure to light (FITC is susceptible to photobleaching)

• Validation approaches:

  • Positive and negative control samples

  • Peptide competition assays

  • Comparison with alternative antibody clones

  • Correlation with other detection methods (e.g., Western blot, ELISA)

To optimize specificity and sensitivity, titrate the antibody concentration and validate using multiple techniques with appropriate controls.

How can researchers troubleshoot weak or non-specific signals when using GAS6 antibodies?

When encountering signal problems with FITC-conjugated GAS6 antibodies, consider the following troubleshooting approaches:

For specific applications like immunohistochemistry of lung tissue, consider specialized approaches such as Sudan Black B treatment to reduce autofluorescence or tyramide signal amplification for enhancing weak signals.

What are the optimal dilution ranges for GAS6 antibody across different applications?

Optimal dilution ranges vary by application and must be empirically determined for each experimental system:

For FITC-conjugated antibodies specifically:

• Start with manufacturer's recommended dilution

• Prepare a dilution series spanning at least 3-fold above and below the recommended concentration

• Evaluate signal-to-noise ratio across the dilution series

• Remember that optimal dilutions should be determined by each laboratory for each application

How can GAS6 antibodies be used to investigate the relationship between GAS6/Axl signaling and lung fibrosis?

GAS6 antibodies offer valuable tools for dissecting the complex role of GAS6/Axl signaling in lung fibrosis:

• Tissue and cellular localization studies:

  • Use FITC-conjugated GAS6 antibodies for immunofluorescence to map GAS6 expression patterns in fibrotic versus normal lung tissue

  • Perform co-localization studies with cell-type markers (e.g., SPC for ATII cells, α-SMA for myofibroblasts)

  • Quantify changes in expression levels and distribution patterns across disease progression

• Signaling pathway analysis:

  • Investigate GAS6-induced TAM receptor activation via phosphorylation status

  • Combine with antibodies against downstream mediators (e.g., COX-2, PGE2, PGD2)

  • Use inhibitors of TAM receptors (e.g., TP-0903) to confirm specificity of pathway involvement

• Functional assays with recombinant GAS6:

  • Monitor migration of lung cells (e.g., H1299 NSCLC cells) with and without rGas6 stimulation

  • Assess EMT marker changes in isolated ATII cells following rGas6 treatment

  • Quantify apoptosis rates in ATII cells after rGas6 administration using TUNEL assays and flow cytometry

• In vivo experimental approaches:

  • Analyze GAS6 protein levels in BAL fluid and conditioned media from primary ATII cells and alveolar macrophages in bleomycin-induced fibrosis models

  • Track changes in EMT markers (E-cadherin, N-cadherin, α-SMA) and apoptosis markers (Bax, cleaved caspase-3, PARP) following rGas6 administration

  • Use double immunofluorescence staining to identify cells undergoing apoptosis and EMT in tissue sections

Recent research has demonstrated that rGas6 administration attenuates lung fibrosis by inhibiting EMT and fibroblast activation, offering a potential therapeutic approach for pulmonary fibrosis .

What methodologies can be employed to study the role of GAS6 in epithelial-mesenchymal transition (EMT)?

EMT is a critical process in fibrosis development, and GAS6's role can be studied using several complementary approaches:

• Morphological and marker analyses:

  • Track morphological changes in isolated ATII cells (from rounded to spindle-shaped) during EMT

  • Quantify epithelial markers (E-cadherin) and mesenchymal markers (N-cadherin, α-SMA) via qRT-PCR, Western blot, and immunofluorescence

  • Monitor EMT transcription factors (Snai1, Zeb1, Twist1) expression levels

• Co-localization studies:

  • Perform double immunofluorescence staining using FITC-conjugated GAS6 antibodies with:

    • EMT markers (E-cadherin/S100A4, α-SMA/S100A4)

    • Cell-type specific markers (SPC for ATII cells)

  • Quantify cells showing co-expression of epithelial and mesenchymal markers

• Functional assays:

  • Cell invasion assays to assess mesenchymal behavior

  • Wound healing assays to evaluate migratory capacity

  • Cell contraction assays to measure myofibroblast function

  • Compare results between control, BLM-treated, and BLM+rGas6-treated groups

• Molecular mechanism investigations:

  • Examine the impact of rGas6 on key EMT regulators using pharmacological inhibitors

  • Evaluate the relationship between GAS6/Axl signaling and COX-2-derived prostaglandin production

  • Use genetic approaches (siRNA, CRISPR) to validate GAS6's role in EMT

These methodologies have revealed that rGas6 administration can reverse the BLM-induced changes in EMT markers, suggesting a protective role against fibrotic transformation .

How can researchers design experiments to distinguish between the roles of different TAM receptors in GAS6 signaling?

GAS6 binds to all three TAM receptors (Tyro3, Axl, and Mer) with different affinities, necessitating careful experimental design to delineate receptor-specific effects:

• Receptor expression profiling:

  • Characterize the expression patterns of all three TAM receptors in the cell/tissue of interest

  • Use immunoblotting, qRT-PCR, and immunofluorescence with receptor-specific antibodies

  • Compare expression levels across different cell types and disease states

• Selective receptor targeting approaches:

  • Use receptor-selective small molecule inhibitors (e.g., TP-0903 for Axl)

  • Apply receptor-specific neutralizing antibodies

  • Employ genetic knockdown/knockout strategies for each receptor individually

  • Utilize receptor-specific siRNAs or shRNAs for transient or stable knockdown

• Receptor activation and signaling studies:

  • Monitor phosphorylation status of each receptor following GAS6 stimulation

  • Track activation of downstream signaling molecules specific to each receptor pathway

  • Use phospho-specific antibodies in Western blot and flow cytometry assays

  • Compare signaling kinetics and dose-response relationships across receptors

• Functional readouts with receptor specificity:

  • Assess cell migration, invasion, and apoptosis protection after selective receptor inhibition

  • Use receptor-expressing cell lines versus receptor-negative controls

  • Implement rescue experiments with receptor re-expression in knockout models

  • Create chimeric receptor constructs to identify domain-specific functions

• Data validation table:

This comprehensive approach has helped researchers determine that GAS6-mediated Axl activation specifically enhances migration of H1299 NSCLC cells, an effect that can be blocked by TP-0903 treatment .

How should researchers interpret contradictory findings regarding GAS6's role in fibrosis across different experimental models?

The literature presents seemingly contradictory findings regarding GAS6's role in fibrosis. A methodical approach to interpreting these discrepancies includes:

• Model-specific considerations:

  • Different fibrosis induction methods (bleomycin, silica, radiation) may activate distinct pathways

  • Timing of intervention is crucial (protective in early inflammation vs. potentially detrimental in established fibrosis)

  • Species differences (mouse vs. human) can affect receptor distribution and signaling outcomes

• Contextual analysis of conflicting data:

  • Genetic deficiency models (Gas6-/- or Mer-/- mice) show protection against silica-induced fibrosis

  • Administration of recombinant GAS6 attenuates bleomycin-induced fibrosis

  • Small molecule TAM inhibitors reduce fibroblast properties in IPF patient samples

  • Protein S (alternative TAM ligand) prevents bleomycin-induced fibrosis

• Reconciliation approaches:

  • Consider biphasic effects depending on disease stage (early vs. late)

  • Examine cell-type specific roles (epithelial protection vs. fibroblast activation)

  • Analyze receptor specificity (Axl vs. Mer signaling may have opposing effects)

  • Evaluate dose-dependent responses (physiological vs. pharmacological levels)

• Experimental design for resolving contradictions:

  • Time-course studies capturing the dynamic changes in GAS6 signaling

  • Cell-type specific conditional knockout approaches

  • Combined inhibition and supplementation experiments

  • Cross-validation across multiple fibrosis models

Research suggests that while GAS6 expression increases in IPF and other fibrotic conditions , exogenous rGas6 administration can paradoxically attenuate fibrosis by preserving epithelial integrity and preventing EMT . This indicates context-dependent functions where timing, concentration, and target cell population significantly influence outcomes.

What methodological approaches can help determine the specificity of observed effects when working with GAS6 antibodies?

Establishing specificity is crucial for confident interpretation of results obtained with GAS6 antibodies:

• Antibody validation controls:

  • Peptide competition/blocking experiments using the immunizing peptide

  • Testing in GAS6 knockout/knockdown systems

  • Comparing results with multiple antibody clones targeting different epitopes

  • Including isotype control antibodies matched to the primary antibody

• Cross-platform verification:

  • Confirm findings using complementary techniques (e.g., if detected by immunofluorescence, verify with Western blot)

  • Use orthogonal approaches (mRNA detection via RT-PCR or RNA-seq)

  • Combine protein detection with functional assays

  • Correlate antibody staining with genetically encoded reporters (e.g., GFP-tagged GAS6)

• Signal specificity assessment:

  • Careful titration of antibody concentration to minimize non-specific binding

  • Implement rigorous background subtraction procedures

  • Use spectral unmixing for autofluorescence in tissues like lung

  • Employ quantitative image analysis with appropriate thresholding

• Biological validation approaches:

  • Demonstrate biological response to GAS6 manipulation (e.g., phosphorylation of Axl after rGas6 treatment)

  • Show expected changes in downstream markers (e.g., EMT markers, apoptosis indicators)

  • Demonstrate reversal of effects with pathway inhibitors (e.g., TP-0903)

  • Correlate antibody-detected expression with expected biological phenomena

These rigorous approaches help ensure that observed effects are genuinely attributable to GAS6, rather than experimental artifacts or cross-reactivity with related proteins.

How can researchers accurately quantify and compare GAS6 expression levels across different experimental conditions?

Accurate quantification of GAS6 expression requires careful standardization and appropriate analytical methods:

• Sample preparation standardization:

  • Establish consistent protocols for tissue/cell processing

  • Standardize protein extraction methods for all samples

  • Use identical fixation parameters for immunofluorescence/IHC

  • Process all experimental conditions in parallel

• Quantitative detection methods:

  • Western blot: Use digital imaging systems with linear dynamic range

  • Immunofluorescence: Apply consistent image acquisition parameters

  • Flow cytometry: Establish proper compensation and utilize fluorescence quantitation beads

  • ELISA: Generate standard curves with recombinant GAS6 protein

• Normalization strategies:

  • Western blot: Normalize to housekeeping proteins (GAPDH) or total protein (Ponceau S)

  • Immunofluorescence: Report signals relative to area, cell number, or specific marker-positive cells

  • qRT-PCR: Use validated reference genes stable across experimental conditions

  • ELISA: Express as absolute concentration based on standard curve

• Statistical analysis requirements:

  • Perform experiments with sufficient biological replicates (n≥3)

  • Apply appropriate statistical tests based on data distribution

  • Report data with measures of central tendency and dispersion

  • Use visualization methods that accurately represent the data distribution

• Quantification workflow example:

For quantifying GAS6 in bleomycin-induced lung fibrosis:

• Collect BAL fluid and conditioned media from primary ATII cells and alveolar macrophages

• Perform Western blot analysis of GAS6 protein levels

• Normalize to appropriate controls

• Compare expression across treatment groups (control, BLM, BLM+rGas6)

• Correlate protein levels with functional readouts (EMT markers, apoptosis indicators)

This approach has revealed that while BLM treatment increases endogenous GAS6 production, additional rGas6 administration does not further enhance endogenous GAS6 levels but still provides protective effects .