GPR153 Antibody

What is GPR153 and why is it significant in neuroscience research?

GPR153 (G Protein-Coupled Receptor 153) is a member of the Class A rhodopsin superfamily of G protein-coupled receptors. This 609 amino acid multi-pass membrane protein functions as an orphan receptor and belongs to the GPR1 family . Its significance in neuroscience research stems from its high expression in central regions of the brain including the thalamus, cerebellum, and arcuate nucleus .

GPR153 has been implicated in several neurological functions and pathologies:

• Knockdown of the orthologous gene in rats is associated with significant reduction in food intake and impaired decision-making ability

• Mutations in GPR153 are associated with schizophrenia, autism, and other neuropsychiatric disorders

• The gene's expression is activated by the glioma-associated oncogene homolog 1 transcription factor, which is activated by sonic hedgehog in normal and tumorigenic cells

What types of GPR153 antibodies are available for research applications?

Several types of validated GPR153 antibodies are available for research applications:

Most commercially available GPR153 antibodies are rabbit polyclonals, with applications primarily in Western blot, immunohistochemistry, immunofluorescence, and ELISA techniques .

How is the specificity of GPR153 antibodies validated?

Validation of GPR153 antibody specificity typically involves multiple complementary approaches:

• Western blot analysis: Detecting a band of approximately 65 kDa (the expected molecular weight of GPR153) in appropriate cell lysates (e.g., K562, Jurkat cells)

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should eliminate or significantly reduce the signal in Western blot and immunofluorescence applications

• Immunohistochemistry validation: Testing on tissue arrays containing multiple normal human tissues (44 normal tissues in the Human Protein Atlas project)

• Independent antibody validation: Comparing staining patterns of multiple antibodies targeting different epitopes of GPR153

• Orthogonal validation: Correlation of protein expression levels with mRNA expression data

Researchers should review validation data provided by manufacturers before selecting an antibody for their specific applications.

What are the optimal protocols for using GPR153 antibodies in immunohistochemistry applications?

For optimal immunohistochemistry (IHC) results with GPR153 antibodies:

Sample Preparation:

• Use 6-μm tissue cryosections or formalin-fixed paraffin-embedded (FFPE) tissue sections

• For FFPE sections, antigen retrieval with TE buffer pH 9.0 is recommended; alternatively, citrate buffer pH 6.0 may be used

Staining Protocol:

• Fix cryosections in cold acetone for 10 minutes at -20°C

• Block with appropriate blocking buffer (typically containing serum from the same species as the secondary antibody)

• Incubate with primary GPR153 antibody at the recommended dilution:

  • For Prestige Antibody HPA007159: 1:50-1:200

  • For Proteintech 24878-1-AP: 1:50-1:500

• Wash thoroughly with PBS (3-5 times)

• Incubate with appropriate secondary antibody (typically anti-rabbit)

• Counterstain nuclei with DAPI

• Mount slides with appropriate mounting medium

For fluorescent detection, use Alexa Fluor-conjugated secondary antibodies. For chromogenic detection, use HRP-conjugated secondaries with appropriate substrate (e.g., DAB).

What controls should be included when performing Western blot analysis with GPR153 antibodies?

Proper experimental controls are critical for ensuring the validity of Western blot results with GPR153 antibodies:

Essential Controls:

• Positive Control: Lysates from cells known to express GPR153, such as:

  • K-562 cells

  • Jurkat cells

  • HeLa cells (for some antibodies)

• Negative Control: One of the following:

  • Peptide competition: Pre-incubation of the antibody with the immunizing peptide

  • Lysates from cells with GPR153 knockdown (siRNA or CRISPR)

  • Tissues or cells known not to express GPR153

• Loading Control: Detection of housekeeping proteins (e.g., GAPDH, β-actin) to ensure equal loading across lanes

• Molecular Weight Marker: To confirm the expected size of approximately 65 kDa for GPR153

Western Blot Protocol Recommendations:

• Dilution range: 1:500-1:1000 for most GPR153 antibodies

• Protein amount: 20-50 μg of total protein per lane

• Detection system: Enhanced chemiluminescence (ECL) or fluorescent-based detection

How can researchers optimize immunofluorescence protocols for GPR153 detection?

For optimal detection of GPR153 by immunofluorescence:

Cell Preparation:

• Fix cells with 4% paraformaldehyde (10-15 minutes at room temperature)

• Permeabilize with 0.1-0.5% Triton X-100 in PBS (5-10 minutes)

• Block with 5% normal serum (from the same species as the secondary antibody) in PBS

Staining Protocol:

• Incubate with primary GPR153 antibody at dilutions of 1:100-1:500

• Wash thoroughly with PBS (3-5 times)

• Incubate with fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488 or 594)

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

Critical Considerations:

• Include a peptide competition control (antibody pre-incubated with immunizing peptide)

• Include a secondary-only control to assess background fluorescence

• GPR153 is a membrane protein, so expect primarily membrane and/or cytoplasmic staining

• Consider co-staining with subcellular markers to confirm localization patterns

How can researchers investigate GPR153 expression in different brain regions?

Investigating GPR153 expression across brain regions requires a strategic approach combining multiple techniques:

Immunohistochemistry Approach:

• Obtain brain tissue sections covering regions of interest (thalamus, cerebellum, arcuate nucleus)

• Perform IHC using validated GPR153 antibodies (e.g., HPA007159)

• Use standardized scoring systems to quantify expression levels

• Compare with expression databases such as the Human Protein Atlas

Comparative Analysis:
Research has shown that GPR153 shares common evolutionary origin with GPR162 and is highly expressed in central brain regions including the thalamus, cerebellum, and the arcuate nucleus . When studying expression patterns, researchers should:

• Design appropriate sampling strategies covering these key regions

• Use serial sections for comprehensive mapping

• Consider co-localization studies with neuronal and glial markers

• Compare findings with published transcriptomic datasets

Technical Considerations:

• For mouse brain studies, perfusion fixation is recommended for optimal tissue preservation

• Consider using tyramide signal amplification for detecting low abundance expression

• For human postmortem tissue, account for postmortem interval effects on immunoreactivity

What approaches can be used to study GPR153 signaling pathways?

As an orphan GPCR, understanding GPR153 signaling requires specialized approaches:

Experimental Strategies:

• G-Protein Coupling Assays:

  • BRET/FRET assays to monitor G-protein activation

  • [35S]GTPγS binding assays to assess G-protein recruitment

  • Second messenger assays (cAMP, Ca2+ mobilization, ERK phosphorylation)

• Receptor Trafficking Studies:

  • Fluorescently tagged GPR153 constructs for live-cell imaging

  • Antibody-based internalization assays

  • Biotinylation assays for surface expression quantification

• Functional Genomics Approaches:

  • CRISPR/Cas9-mediated knockout or knockin models

  • Conditional expression systems for temporal control

  • Single-cell transcriptomics to identify downstream gene targets

Data Interpretation Considerations:

• As an orphan receptor, basal activity may be significant

• Consider constitutive activity parameters in experimental design

• Compare with closely related GPCRs such as GPR162

How can GPR153 function be studied in relation to neuropsychiatric disorders?

Given the association of GPR153 mutations with neuropsychiatric disorders , researchers can employ several approaches:

Translational Research Strategies:

• Genetic Association Studies:

  • Screen for GPR153 mutations in patient cohorts with schizophrenia, autism, etc.

  • Perform functional characterization of identified variants using in vitro assays

• Animal Models:

  • Generate GPR153 knockout or knockin mice modeling human mutations

  • Assess behavioral phenotypes relevant to neuropsychiatric disorders

  • Evaluate pharmacological rescue strategies

• Induced Pluripotent Stem Cell (iPSC) Models:

  • Generate iPSCs from patients with GPR153 mutations

  • Differentiate into neurons for functional studies

  • Perform drug screening for potential therapeutic compounds

Experimental Design Considerations:

• Include appropriate control groups (both wild-type and heterozygous models)

• Use standardized behavioral testing batteries

• Consider developmental timing of GPR153 expression in experimental design

• Implement both cellular and systems-level analyses

What are common issues encountered with GPR153 antibodies and how can they be resolved?

Researchers working with GPR153 antibodies frequently encounter several technical challenges:

Validation Strategies:
When troubleshooting, consider additional validation approaches:

• Compare multiple antibodies targeting different epitopes of GPR153

• Include appropriate positive and negative controls

• Correlate protein detection with mRNA expression data

How can researchers differentiate between GPR153 and related GPCRs in their experiments?

Distinguishing GPR153 from related GPCRs requires careful experimental design:

Specificity Strategies:

• Antibody Selection:

  • Choose antibodies raised against unique regions of GPR153

  • Review the immunogen sequence to assess potential cross-reactivity

  • For example, HPA007159 targets a unique sequence: YRADLKAVREKCMALMANDEESDDETSLEGGISPDLVLERSLDYGYGGDFVALDRMAKYEISALEGGLPQLYPLRPLQEDKMQYLQVPPTRRFSHDDADVW

• Control Experiments:

  • Include GPR153 knockout or knockdown samples

  • Perform competitive binding with immunizing peptide

  • Consider testing in cell lines with known expression profiles of related GPCRs

• Combined Detection Methods:

  • Correlate protein detection with mRNA expression using specific primers

  • Consider RNAscope assays for high-specificity mRNA detection

  • Use epitope-tagged constructs for exogenous expression studies

Key Related GPCRs to Consider:

• GPR162: Shares evolutionary origin with GPR153

• GPR151: Another orphan GPCR with partial sequence similarity

• Other members of the GPR1 family

What methodological approaches are recommended for studying GPR153 in tissue microarrays?

Tissue microarray (TMA) analysis of GPR153 requires specific methodological considerations:

Optimized Protocol:

• TMA Construction:

  • Include tissues with known GPR153 expression (e.g., brain regions) as positive controls

  • Include tissues with minimal expression as negative controls

  • Use multiple cores per tissue type to account for heterogeneity

• Staining Optimization:

  • Perform antigen retrieval optimization (test both citrate buffer pH 6.0 and TE buffer pH 9.0)

  • Titrate antibody dilution (starting with manufacturer recommendations)

  • Consider signal amplification for low-abundance detection

• Scoring and Analysis:

  • Implement standardized scoring systems (e.g., H-score, Allred score)

  • Consider automated image analysis for quantification

  • Document subcellular localization patterns

Quality Control Measures:

• Include tissue orientation markers

• Process all TMA slides in a single batch to minimize variability

• Perform replicate staining to ensure reproducibility

• Include isotype control antibodies to assess background

The Human Protein Atlas project has extensively validated GPR153 antibodies in tissue arrays containing 44 normal human tissues and 20 of the most common cancer tissues . Researchers can reference these datasets when designing their TMA experiments.

How can new technological advances enhance GPR153 antibody-based research?

Emerging technologies are expanding the capabilities of GPR153 antibody-based research:

Advanced Imaging Approaches:

• Super-resolution microscopy (STORM, PALM) for nanoscale localization of GPR153

• Expansion microscopy for improved spatial resolution in complex tissues

• Multiplex immunofluorescence for co-expression analysis with up to 40 markers

Single-Cell Analysis:

• Mass cytometry (CyTOF) with GPR153 antibodies for high-dimensional analysis

• Spatial transcriptomics coupled with protein detection

• Imaging mass cytometry for tissue-context protein mapping

Automation and High-Throughput Screening:

• Automated immunostaining platforms for reproducibility

• High-content screening of GPR153 modulators

• AI-assisted image analysis for complex expression pattern recognition

These technological advances will enable more comprehensive characterization of GPR153 expression and function in normal physiology and disease states.

What are promising research directions for understanding GPR153 function in health and disease?

Based on current knowledge, several promising research directions for GPR153 include:

• Identification of Endogenous Ligands:

  • GPR153 is currently classified as an orphan receptor

  • Screening approaches including functional assays and computational predictions may identify natural ligands

  • Understanding of signaling mechanisms would be greatly enhanced by ligand identification

• Neural Circuit Function:

  • Given high expression in key brain regions , investigation of GPR153's role in specific neural circuits

  • Potential implications for feeding behavior based on knockdown studies

  • Circuit-specific conditional knockout models may reveal region-specific functions

• Psychiatric Disorder Mechanisms:

  • Further characterization of how GPR153 mutations contribute to schizophrenia and autism

  • Development of small molecule modulators as potential therapeutic agents

  • Integration with broader neuropsychiatric genetic networks

• Cross-Talk with Other GPCR Systems:

  • Investigation of potential dimerization with other GPCRs

  • Shared signaling pathways with related receptors like GPR162

  • Comparative analysis with other GPCRs implicated in neuropsychiatric disorders

These directions represent valuable opportunities for researchers to advance understanding of this understudied but potentially important GPCR.