GPR116 Antibody

What is GPR116 and why is it important in research?

GPR116 (also known as ADGRF5, adhesion G protein-coupled receptor F5) is a member of the adhesion G protein-coupled receptor family involved in cell surface receptor signaling pathways. It plays critical roles in surfactant homeostasis in lungs, glomerular filtration in kidneys, and pharyngeal arch artery morphogenesis . It has gained significant research interest due to its emerging roles in cancer progression and its tissue-specific functions. Detecting GPR116 with antibodies is crucial for understanding its expression patterns and physiological roles.

What applications are GPR116 antibodies validated for?

GPR116 antibodies are validated for multiple applications including:

Note: Optimal dilutions should be determined by each laboratory for their specific application .

How do I select the appropriate GPR116 antibody for my research?

Selection should be based on:

• Species reactivity - confirm the antibody recognizes your target species (human GPR116 antibodies show cross-reactivity with primate samples and predicted reactivity with mouse, rat, and other mammals at varying homology percentages)

• Application compatibility - ensure validation for your intended application (WB, IHC, flow cytometry)

• Epitope location - antibodies targeting different domains (extracellular, transmembrane, or cytoplasmic) may yield different results

• Clonality - polyclonal antibodies offer broader epitope recognition while monoclonal antibodies provide higher specificity

What is the expression pattern of GPR116 across different tissues?

GPR116 shows differential expression across tissues:

This expression pattern has been confirmed at the protein level by Western blot analysis .

How is GPR116 localized in kidney cells and what methodology best detects this?

GPR116 shows specific cellular localization in the kidney:

• Primarily expressed in acid-secreting A-intercalated cells (A-ICs) in the cortical and medullary collecting ducts

• Colocalization with the B subunit of vacuolar-type H+-ATPase in the medulla

• In outer medullary collecting ducts, GPR116-expressing cells are interspersed among AQP2-expressing principal cells

• In the renal cortex, GPR116 localizes to only a subset of AQP2-negative cells

For optimal detection of this pattern, immunofluorescence imaging on fixed-frozen kidney sections using validated antibodies is recommended. Colocalization studies with markers like V-ATPase (for A-ICs) and AQP2 (for principal cells) provide definitive cellular identification .

What are the recommended protocols for using GPR116 antibody in Western blot analysis?

For optimal Western blot detection of GPR116:

• Sample preparation:

  • Use whole-organ lysates or cell lysates (HEK293, HeLa, or Y79 cells show positive expression)

  • Include positive controls (e.g., HEK293 cells expressing murine or human GPR116)

• Gel electrophoresis:

  • Use appropriate percentage gels to resolve the 149 kDa protein

  • Load adequate protein amount (30-50 μg total protein)

• Transfer and detection:

  • Use PVDF membrane for optimal protein binding

  • Block with 5% non-fat milk or BSA

  • Apply primary antibody at 1:500-1:1000 dilution

  • Incubate overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibody

• Expected results:

  • Observed band at approximately 149 kDa (calculated molecular weight)

  • Validation in lung, kidney, and heart tissues as positive controls

What antigen retrieval methods are recommended for GPR116 immunohistochemistry?

For successful GPR116 immunohistochemistry:

• Primary antigen retrieval methods:

  • TE buffer pH 9.0 (recommended as first choice)

  • Citrate buffer pH 6.0 (alternative method)

• Protocol specifics:

  • For FFPE tissue sections: 5-7 μm thickness

  • Heat-induced epitope retrieval: 95-98°C for 15-20 minutes

  • Cool to room temperature: 20 minutes

  • Block endogenous peroxidase activity: 3% H₂O₂

  • Block non-specific binding: 5-10% normal serum

  • Primary antibody incubation: 1:50-1:500 dilution, overnight at 4°C

  • Detection system: HRP-polymer or ABC method with DAB visualization

• Special considerations:

  • Human kidney tissue serves as positive control

  • Brain (substantia nigra, neurons) and breast carcinoma tissue also show positive staining

How can GPR116 antibodies be used to investigate receptor activation mechanisms?

Investigating GPR116 activation requires specialized approaches:

• Tethered agonist activation studies:

  • Generate constructs with mutations near GPS cleavage site (e.g., H991A mutation)

  • Use antibodies to detect receptor cleavage products (NTF and CTF)

  • Compare wild-type and mutant receptors using conformational antibodies

• Molecular mechanism analysis:

  • Employ antibodies recognizing specific domains to monitor conformational changes

  • Detect interactions with downstream signaling proteins (GNAQ/GNA11)

  • Track receptor internalization upon activation

• In vivo validation using knock-out models:

  • Use antibodies to confirm deletion or mutation of GPR116

  • Assess expression of downstream targets in conditional knock-out tissues

  • Monitor phenotypic changes (e.g., surfactant accumulation)

How can GPR116 antibodies be used to establish its role as a prognostic biomarker in cancer?

For cancer prognostic studies:

• Tissue microarray analysis:

  • Optimize antibody dilution (1:50-1:500) for IHC on FFPE cancer tissues

  • Establish scoring system (e.g., intensity score 0-3+)

  • Correlate staining intensity with clinicopathological parameters

• Survival analysis methodology:

  • Stratify patients based on GPR116 expression levels

  • Generate Kaplan-Meier survival curves based on antibody staining intensity

  • Perform multivariate Cox regression analysis to establish independent prognostic value

• Example findings from gastric cancer studies:

  • High GPR116 expression correlated with advanced TNM stage

  • Multivariate analysis showed GPR116 overexpression as an independent prognostic indicator (HR = 1.855, 95% CI 1.021–3.370, P = 0.043)

  • ROC analysis demonstrated predictive value with area under curve of 0.8104

What are common issues when using GPR116 antibodies and how can they be resolved?

Common challenges and solutions:

• High background in IHC:

  • Increase blocking time/concentration (use 5-10% normal serum)

  • Optimize antibody dilution (try 1:100 instead of 1:50)

  • Include additional washing steps

  • Test alternative antigen retrieval methods (switch from TE pH 9.0 to citrate pH 6.0)

• Weak or no signal in Western blot:

  • Ensure adequate protein loading (≥30 μg)

  • Optimize transfer conditions for high molecular weight protein (149 kDa)

  • Increase antibody concentration or incubation time

  • Use enhanced chemiluminescence detection system

• Non-specific bands:

  • Increase antibody dilution (1:1000 instead of 1:500)

  • Use highly pure samples

  • Include appropriate controls (knockout tissue/cells)

How can GPR116 antibodies be validated for specificity in research applications?

Thorough validation approaches include:

• Genetic controls:

  • Compare staining in wild-type vs. GPR116 knockout tissues

  • Use siRNA/shRNA knockdown cells versus control cells

  • Test over-expression systems (e.g., HEK293 cells transfected with GPR116)

• Epitope-specific validation:

  • Use blocking peptides specific to the antibody epitope

  • Compare antibodies targeting different domains of GPR116

  • Analyze GPR116 mutants (e.g., Δexon17 Gpr116) to confirm specificity

• Cross-reactivity assessment:

  • BLAST analysis of immunogen sequence (e.g., one GPR116 antibody immunogen showed no homology with other human proteins except KLHL24 at 50%)

  • Test in multiple species according to predicted homology

  • Verify using orthogonal methods (mass spectrometry, RNA-seq correlation)

What signaling pathways are activated downstream of GPR116 and how can these be monitored?

GPR116 activates several signaling pathways that can be monitored using antibody-based techniques:

• Primary signaling pathways:

  • PI3K-Akt signaling pathway

  • Extracellular matrix-receptor interaction

  • Focal adhesion signaling

  • Cell adhesion pathways

• Detection methods:

  • Phospho-specific antibodies to track Akt activation

  • Co-immunoprecipitation to detect GPR116 interaction with GNAQ/GNA11

  • Western blotting for downstream signaling intermediates

  • Immunofluorescence to track protein translocation upon activation

• Functional correlation:

  • Gene expression changes using RT-PCR or RNA-seq

  • Connect signaling activation to physiological responses (e.g., surfactant secretion)

How can GPR116 antibodies be used to investigate its role in pulmonary surfactant regulation?

Investigating GPR116 in surfactant regulation:

• Cellular localization studies:

  • Use antibodies to confirm GPR116 expression in alveolar type II epithelial (AT2) cells

  • Perform immunofluorescence co-staining with AT2 cell markers

• Functional studies:

  • Compare GPR116 expression in normal vs. surfactant-accumulation conditions

  • Analyze effects of Δexon17 GPR116 mutation using domain-specific antibodies

  • Monitor localization changes in response to surfactant stimulation

• Mechanistic investigation:

  • Assess GPS cleavage site mutations (e.g., H991A) using antibodies recognizing cleaved forms

  • Track receptor activation using phospho-specific antibodies

  • Correlate GPR116 signaling with surfactant protein expression and secretion

How are GPR116 antibodies being used to explore its potential as a therapeutic target?

Emerging therapeutic targeting approaches:

• Expression profiling in disease states:

  • Use antibodies to quantify GPR116 upregulation in cancer tissues

  • Compare expression levels between primary tumors and metastases

  • Correlate with treatment response in patient cohorts

• Mechanistic studies for drug development:

  • Screen for compounds that modulate GPR116 activity using antibody-based assays

  • Detect conformational changes induced by potential therapeutic agents

  • Monitor downstream signaling modulation using phospho-specific antibodies

• Therapeutic antibody development:

  • Generate function-blocking antibodies targeting extracellular domains

  • Test effects on cancer cell migration and invasion

  • Develop antibody-drug conjugates targeting GPR116-positive tumors

What is the current understanding of GPR116 mutations and how can antibodies detect variant forms?

Current knowledge and detection methods:

• Known GPR116 variants:

  • Exon17 deletion affects transmembrane domains

  • H991A mutation prevents GPS autocatalytic cleavage

  • Various splice variants may exist across tissues

• Detection strategies:

  • Domain-specific antibodies to distinguish variant forms

  • Western blotting to assess molecular weight differences

  • Immunoprecipitation followed by mass spectrometry for detailed characterization

• Functional implications:

  • Δexon17 mutation leads to protein mislocalization (cytoplasmic vs. membrane)

  • Differential antibody reactivity with cleaved vs. uncleaved forms

  • Altered signaling capacity of variant forms can be assessed using phospho-specific antibodies