GPR35 Antibody

What is GPR35 and where is it expressed in human tissues?

GPR35 is a G protein-coupled receptor that plays dual roles in modulating inflammatory responses. It is highly expressed in the gastrointestinal tract, predominantly in colon epithelial cells, and is also found in various immune cells . GPR35 binds to several ligands including the tryptophan metabolite kynurenic acid (KYNA), lysophosphatidic acid (LPA), and 5-hydroxyindoleacetic acid (5-HIAA) . The receptor mediates rapid and transient activation of numerous intracellular signaling pathways.

Expression pattern by tissue type:

• High expression: Colon, ileum, intestinal epithelial cells

• Moderate expression: Immune cells (neutrophils, macrophages)

• Detected in: Pancreas, kidney, lung, spleen, and muscle

When designing experiments targeting GPR35, consider its abundance in intestinal tissues for optimal detection using validated antibodies .

What are the main isoforms of GPR35 and how do they differ?

GPR35 exists as two distinct isoforms that emerge from different promoter usage and alternative splicing:

These isoforms share similar cellular localization but demonstrate different signaling properties . The extended N-terminus of GPR35b limits G protein activation while elevating receptor-β-arrestin interaction . When selecting antibodies, consider whether your research requires detection of both isoforms or specific targeting of one variant.

What experimental applications are GPR35 antibodies validated for?

Based on the search results, GPR35 antibodies have been validated for the following applications:

For optimal results, use antibodies that have been specifically validated for your application of interest and include appropriate positive controls such as human ileum tissue or MCF-7 cells, which have been confirmed to express GPR35 .

How do different experimental systems affect GPR35 detection and signaling analysis?

GPR35 signaling can be studied using multiple experimental systems, each with distinct advantages and limitations:

When selecting antibodies for these assays, consider epitope accessibility in your experimental system. For instance, N-terminal tag antibodies may be ineffective for internalization studies if the N-terminus becomes inaccessible during conformational changes .

How can researchers optimize Western blot protocols for GPR35 detection?

Optimizing Western blot protocols for GPR35 detection requires attention to several key factors:

• Sample preparation:

  • Use RIPA or modified RIPA buffer with protease inhibitors

  • For membrane proteins like GPR35, include membrane solubilization steps

  • Run under reducing conditions using Immunoblot Buffer Group 1

• Gel selection and transfer:

  • Use PVDF membrane (preferred over nitrocellulose for GPR35)

  • 10-12% gels provide optimal separation for the 34 kDa GPR35 protein

• Antibody conditions:

  • Primary antibody concentration: 2 μg/mL has been validated

  • Secondary antibody: HRP-conjugated anti-species antibody (e.g., HAF018)

  • Include blocking with 5% non-fat milk or BSA

• Positive controls:

  • Human ileum tissue lysate

  • MCF-7 human breast cancer cell line

Expected outcome: A specific band should be detected at approximately 34 kDa for GPR35a (the predominant isoform) .

What strategies can be employed to study GPR35 disease-associated variants?

GPR35 has been implicated in inflammatory bowel diseases (IBD) through genome-wide association studies, with several SNPs identified as risk factors . When studying these variants:

• Key disease-associated variants to consider:

  • rs3749171 (T108M) - Most extensively studied, causes a threonine to methionine transition

  • rs4676410 - Upstream intron variant with higher prevalence

• Experimental approaches:

  • Use CRISPR/Cas9 gene editing to create cell lines with specific variants

  • Compare signaling responses between wild-type and variant receptors using:

    • β-arrestin recruitment assays

    • G-protein activation assays

    • Downstream signaling (ERK1/2 phosphorylation)

• Antibody considerations:

  • Verify the epitope location relative to the variant position

  • For T108M variant, use antibodies targeting regions unaffected by the mutation

  • Consider using isoform-specific antibodies if the variant affects splicing

Research has shown that the T108M variant is hypermorphic, leading to hyperactivation of GPR35 with increased proliferation and metabolism in intestinal epithelial cells and bone marrow-derived macrophages .

How should researchers design experiments to investigate GPR35 function in inflammation?

When investigating GPR35's role in inflammation, consider the following experimental design:

• Cell type selection:

  • Intestinal epithelial cells (high endogenous expression)

  • Macrophages (GPR35 mediates TNF responses)

  • Neutrophils (GPR35 mediates recruitment to inflammatory sites)

• Functional assays:

  • Cytokine production: Measure IL-8, VEGF, TNF following GPR35 activation/inhibition

  • Cell migration: Assess GPR35's role in chemotaxis of immune cells

  • Barrier function: Evaluate epithelial integrity through transepithelial resistance

• Antibody application strategies:

  • Use blocking antibodies to inhibit GPR35 function

  • Employ antibodies for immunoprecipitation to identify binding partners

  • Apply phospho-specific antibodies to track downstream signaling events

• In vivo models:

  • GPR35-deficient mice show reduced susceptibility to ETBF-induced colitis

  • GPR35 is required for goblet cell function and protection against certain bacterial infections

Remember that GPR35's role can be context-dependent, showing pro-inflammatory effects in some settings and anti-inflammatory effects in others .

What are the critical controls needed when validating a new GPR35 antibody?

Thorough validation of GPR35 antibodies requires multiple controls:

• Positive tissue/cell controls:

  • Human ileum tissue (verified high expression)

  • MCF-7 cells (confirmed expression)

  • A549 cells (validated for flow cytometry)

• Negative controls:

  • GPR35 knockout cells (CRISPR/Cas9-generated)

  • Non-expressing cell lines

  • Secondary antibody-only controls

• Specificity controls:

  • Peptide competition assays using the immunizing peptide

  • siRNA or shRNA knockdown of GPR35

  • Overexpression systems comparing empty vector vs. GPR35-expressing cells

• Cross-reactivity assessment:

  • Test against related GPCRs to ensure specificity

  • If studying orthologs, verify species cross-reactivity (human GPR35 shares 73% homology with mouse GPR35)

Document antibody validation experiments thoroughly, including images of western blots showing specific bands at the expected molecular weight (~34 kDa) and appropriate controls.

How can researchers distinguish between GPR35 isoforms in experimental systems?

Distinguishing between GPR35 isoforms requires strategic experimental approaches:

• Antibody selection:

  • Use antibodies targeting the N-terminal extension unique to GPR35b

  • For GPR35a-specific detection, select antibodies against epitopes absent in GPR35b

• Molecular techniques:

  • Design PCR primers spanning the alternative splice junctions

  • Use isoform-specific siRNAs for selective knockdown

• Expression profiling:

  • Conduct promoter analysis to understand differential expression

  • Analyze tissue-specific expression patterns (GPR35b is enriched in cancer cells)

• Functional discrimination:

  • Compare G-protein vs. β-arrestin signaling (GPR35b shows elevated β-arrestin interaction)

  • Assess receptor internalization kinetics, which may differ between isoforms

• Molecular weight discrimination:

  • Use high-resolution gel systems to separate the isoforms by size

  • GPR35a runs at ~34 kDa while GPR35b appears at ~38 kDa

Research has shown these isoforms emerge from distinct promoter usage and have functional differences despite similar cellular localization .

What optimization strategies are recommended for flow cytometry using GPR35 antibodies?

Flow cytometry with GPR35 antibodies requires careful optimization:

• Sample preparation considerations:

  • For intracellular staining: fix cells with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 or commercial permeabilization buffers

  • For surface staining: use gentle fixation to preserve epitope integrity

• Antibody titration:

  • Start with 0.40 μg per 10^6 cells in 100 μl suspension

  • Create a titration curve to determine optimal signal-to-noise ratio

  • Test multiple antibody concentrations and incubation times

• Compensation controls:

  • Single-color controls for each fluorophore

  • FMO (Fluorescence Minus One) controls

  • Isotype controls matched to antibody class and species

• Gating strategy:

  • Exclude debris and doublets

  • Use viability dye to remove dead cells

  • Establish positive population based on appropriate controls

• Fluorophore selection:

  • For direct conjugates, CoraLite® Plus 647 has been validated (Excitation/Emission: 654nm/674nm)

  • Consider brightness requirements based on expression level

When using conjugated antibodies, verify they maintain specificity after conjugation and store protected from light at appropriate temperatures (-20°C) .

How should researchers interpret conflicting GPR35 data across different experimental systems?

When facing conflicting GPR35 data, consider these systematic evaluation approaches:

• Species-specific differences:

  • Human GPR35 shares only 73% homology with mouse GPR35

  • Zebrafish has two paralogs with only ~26% and 24% similarity to human GPR35

  • Pharmacological properties differ substantially between species orthologs

• Isoform variations:

  • GPR35a and GPR35b have distinct signaling properties

  • The extended N-terminus of GPR35b limits G protein activation but enhances β-arrestin recruitment

• Signaling pathway divergence:

  • GPR35 couples to multiple G proteins (Gα12/13, Gαi/o)

  • Different assay systems may preferentially detect certain pathways

  • Context-dependent signaling based on cell type and environment

• Methodological considerations:

  • Assay sensitivity differences (BRET vs. immunoassays)

  • Expression levels affecting signal detection

  • Buffer conditions influencing receptor conformation

To reconcile conflicting data, conduct parallel experiments using standardized conditions and multiple detection methods to establish consistency across platforms.

What are the common pitfalls in GPR35 antibody-based research and how can they be avoided?

Common pitfalls in GPR35 antibody research include:

• Antibody specificity issues:

  • Problem: Cross-reactivity with related GPCRs

  • Solution: Validate using GPR35 knockout controls and peptide competition assays

• Post-translational modifications affecting detection:

  • Problem: Glycosylation altering apparent molecular weight

  • Solution: Include deglycosylation steps in sample preparation when appropriate

• Isoform confusion:

  • Problem: Antibodies detecting both isoforms without discrimination

  • Solution: Use isoform-specific antibodies or complement with PCR-based detection

• Buffer compatibility:

  • Problem: Membrane protein solubilization issues

  • Solution: Optimize lysis conditions for GPCRs (mild detergents, physiological pH)

• Signal interpretation challenges:

  • Problem: Distinguishing specific from non-specific bands

  • Solution: Include appropriate positive controls (ileum tissue, MCF-7 cells)

• Reproducibility issues:

  • Problem: Inconsistent results between experiments

  • Solution: Standardize protocols and document all experimental variables

Always report comprehensive methodological details, including antibody catalog numbers, dilutions, incubation conditions, and detailed sample preparation steps to enhance reproducibility.

How can researchers effectively combine antibody-based detection with functional GPR35 assays?

Integrating antibody detection with functional assays provides comprehensive insights into GPR35 biology:

• Sequential analysis workflow:

  • Confirm GPR35 expression via Western blot or flow cytometry

  • Perform immunofluorescence to determine subcellular localization

  • Conduct functional assays (signaling, trafficking, cellular responses)

  • Correlate expression levels with functional outcomes

• Complementary assay combinations:

  Antibody Technique	Complementary Functional Assay	Insight Gained
  Western blot	β-arrestin recruitment BRET	Correlation between expression and signaling capacity
  Flow cytometry	G-protein activation ([35S]GTPγS)	Relationship between receptor levels and G-protein coupling
  Immunofluorescence	Receptor internalization	Trafficking dynamics and subcellular localization
  Immunoprecipitation	Mass spectrometry	Identification of binding partners

• Validation strategies:

  • Use GPR35 agonists (zaprinast, pamoic acid) to confirm functional responses

  • Apply GPR35 antagonists (ML145) to block responses and confirm specificity

  • Compare wild-type vs. mutant (T108M) responses to understand variant effects

• Data integration approaches:

  • Normalize functional responses to expression levels

  • Perform correlation analyses between expression and functional readouts

  • Consider both spatial (localization) and temporal (kinetics) aspects

This integrated approach provides stronger evidence for GPR35-specific effects than either antibody detection or functional assays alone.

How can GPR35 antibodies contribute to understanding the receptor's role in inflammatory bowel disease?

GPR35 antibodies are valuable tools for investigating this receptor's role in IBD pathogenesis:

• Tissue expression profiling:

  • Compare GPR35 expression in healthy vs. IBD patient samples

  • Analyze expression changes during disease progression

  • Correlate expression with clinical parameters

• Cellular distribution analysis:

  • Determine which immune cell populations express GPR35 in IBD

  • Assess epithelial vs. immune cell expression patterns

  • Examine changes in subcellular localization during inflammation

• Genetic variant investigation:

  • Develop antibodies specifically recognizing the T108M variant

  • Compare signaling properties of wild-type vs. variant receptors

  • Assess how variants affect interaction with binding partners

• Therapeutic targeting validation:

  • Use antibodies to confirm target engagement of GPR35-directed drugs

  • Develop blocking antibodies as potential therapeutic agents

  • Assess receptor modulation following treatment interventions

Research has established that GPR35 risk variants are associated with IBD, with the T108M variant showing hyperactivation that leads to increased proliferation and altered immune responses . GPR35-deficient mice demonstrate altered susceptibility to bacterial infections, highlighting its complex role in intestinal homeostasis .

What novel techniques are being developed for studying GPR35 receptor dynamics and signaling?

Emerging techniques for GPR35 research include:

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize receptor nanoclusters

  • Single-molecule tracking to follow receptor movement in real-time

  • FRET/BRET biosensors for conformational changes and protein interactions

• Structural biology tools:

  • Cryo-EM to determine GPR35 structure in different activation states

  • HDX-MS (hydrogen-deuterium exchange mass spectrometry) to study conformational dynamics

  • Computational modeling to predict ligand binding and receptor activation

• Genetic manipulation techniques:

  • CRISPR-based approaches for endogenous tagging of GPR35

  • Optogenetic control of GPR35 signaling

  • Knock-in models of disease-associated variants

• Multiplexed analysis systems:

  • Single-cell analysis combining expression and functional readouts

  • Phosphoproteomics to map GPR35 signaling networks

  • Biosensor arrays for parallel monitoring of multiple signaling pathways

These techniques, when combined with well-validated antibodies, will provide unprecedented insights into GPR35 biology and facilitate development of targeted therapeutics for inflammatory conditions.