GPR162 Antibody

What is GPR162 and why is it important for research?

GPR162 (G Protein-Coupled Receptor 162) is a member of the G protein-coupled receptor family, which comprises transmembrane proteins involved in signal transduction. These receptors are crucial for various physiological processes and represent significant therapeutic targets. GPR162 specifically appears to be a neuronal orphan GPCR, meaning its endogenous ligand remains unidentified. Research suggests it may participate in ERK signaling pathways, particularly in neuronal cells, which makes it relevant for neuroscience research . Understanding GPR162's function could provide insights into neuronal signaling mechanisms and potentially reveal new therapeutic targets for neurological conditions. The molecular characterization of GPR162 is ongoing, with current research focusing on its expression patterns and potential involvement in cellular signaling cascades.

What types of GPR162 antibodies are available for research applications?

Several types of GPR162 antibodies are available for research, primarily polyclonal antibodies raised in rabbits. These antibodies target different domains of the GPR162 protein, including:

This diversity allows researchers to select antibodies specific to their experimental needs, target species, and applications . The availability of antibodies targeting different epitopes is particularly valuable for validation studies, as it allows confirmation of results using multiple antibodies recognizing different regions of the same protein.

What are the common applications for GPR162 antibodies?

GPR162 antibodies can be utilized in multiple research applications based on the available data:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of GPR162 in solution .

• Western Blotting (WB): For protein detection and semi-quantitative analysis, typically using dilutions of 1/500 to 1/2000 .

• Immunohistochemistry (IHC): For localization studies in tissue sections .

• Immunocytochemistry (ICC): For cellular localization studies with certain antibodies .

• Immunofluorescence (IF): Both cellular (cc) and paraffin-embedded (p) applications are possible with select antibodies .

Each application requires specific optimization, and researchers should consider factors such as antibody concentration, incubation conditions, and detection systems based on their experimental design. The recommended dilutions provided by manufacturers serve as starting points for optimization in individual laboratory settings.

How can GPR162 antibodies be used to study GPCR signaling pathways?

GPR162 antibodies can be instrumental in investigating GPCR signaling cascades through several methodological approaches. Research indicates that GPR162, as an orphan GPCR, may influence ERK and Akt phosphorylation pathways . To study these signaling mechanisms, researchers can:

• Combine GPR162 antibodies with phospho-specific antibodies (p-ERK, p-Akt) in western blotting to correlate GPR162 expression with downstream signaling activation .

• Use immunoprecipitation with GPR162 antibodies followed by mass spectrometry to identify interacting partners within signaling complexes.

• Implement immunofluorescence co-localization studies to visualize the spatial relationship between GPR162 and other signaling components.

When studying overexpression systems, researchers have observed significant increases in ERK and Akt phosphorylation in both HEK-293T and N2A neuronal cells expressing GPCRs including GPR1, GPR19, GPR21, and GPR61 . Similar methodologies could be applied to study GPR162's signaling mechanisms. Quantification of these phosphorylation events provides measurable outcomes for assessing the functional impact of GPR162 in cellular signaling networks.

What are the challenges in detecting endogenous GPR162 in different tissue types?

Detecting endogenous GPR162 presents several methodological challenges that researchers must address:

• Expression level variation: GPR162 may be expressed at low levels in certain tissues, requiring sensitive detection methods. Researchers should consider signal amplification techniques for IHC applications.

• Specificity concerns: Given the structural similarities among GPCRs, antibody cross-reactivity must be rigorously controlled through:

  • Inclusion of appropriate negative controls (tissues from knockout models)

  • Validation with multiple antibodies targeting different epitopes

  • Correlation with mRNA expression data from PCR studies

• Technical considerations for different tissue types:

  • Neural tissues may require specialized fixation protocols to preserve GPR162 antigenicity

  • Membrane protein extraction efficiency varies between tissue types and requires optimization

  • Background autofluorescence in certain tissues may interfere with immunofluorescence detection

The observed molecular weight of GPR162 appears to vary (33 kDa and 64 kDa observed in some studies) , suggesting possible post-translational modifications or alternative splicing that may complicate detection and interpretation. Researchers should validate any unexpected molecular weight observations with additional molecular techniques.

How can shRNA knockdown approaches be optimized to study GPR162 function?

Optimizing shRNA knockdown for GPR162 functional studies requires careful methodological consideration. Based on the available research, a systematic approach includes:

• Design and selection of effective shRNA sequences:

  • The sequence 5'-GGCACCTGTGACGACTAC-3' has been used successfully for GPR162 targeting

  • Multiple shRNA constructs should be tested to identify those with highest knockdown efficiency

  • Non-targeting scrambled sequences should be included as controls

• Delivery optimization:

  • Lentiviral systems have been effective, with packaging vectors pMD2.G, pRSV-Rev, and pMDLg/pRRE

  • Viral titer determination is critical, with approximately 2×10^5 TDU (transduction units) recommended for 2×10^5 cells

  • Validation of transduction efficiency using reporter genes (e.g., GFP) in parallel constructs

• Knockdown verification:

  • qRT-PCR using validated primers (forward: 5'-CTCGTCGGGAGTGCGTCT-3', reverse: 5'-GTGGGTGTCTTGGTGCACAG-3')

  • Western blot confirmation of protein reduction

  • Normalization to appropriate housekeeping genes (GAPDH for mRNA studies)

• Functional assessment:

  • Analyzing changes in ERK and Akt phosphorylation levels following stimulation

  • Complementation studies with shRNA-resistant constructs to confirm specificity

Statistical analysis using appropriate tests (e.g., Student's t-test for two-group comparisons or ANOVA with Bonferroni's or Dunnett's post-hoc tests for multiple comparisons) ensures proper interpretation of knockdown effects on signaling pathways .

What optimization strategies are recommended for Western blot detection of GPR162?

Optimizing Western blot protocols for GPR162 detection requires addressing several critical parameters:

• Sample preparation considerations:

  • Effective membrane protein extraction is crucial since GPR162 is a transmembrane protein

  • Sample buffers containing adequate detergents (e.g., Triton X-100 or CHAPS) facilitate solubilization

  • Protease inhibitors must be included to prevent degradation

• Antibody selection and dilution:

  • For GPR162, recommended dilutions typically range from 1/500 to 1/2000 for Western blotting

  • Primary antibody incubation is often optimal at 4°C overnight to maximize specific binding

  • Secondary antibody selection should match the host species (typically anti-rabbit for GPR162 antibodies)

• Detection considerations:

  • Expected molecular weights of 33 kDa and/or 64 kDa have been reported

  • Multiple bands may indicate post-translational modifications or alternative splicing

  • Positive controls (overexpression lysates) can help validate band identity

• Troubleshooting common issues:

  • High background: Increase blocking time/concentration or add Tween-20 to washing buffers

  • No signal: Verify protein transfer efficiency with reversible stains; consider increasing antibody concentration

  • Multiple non-specific bands: Increase antibody dilution or implement more stringent washing conditions

For quantitative analysis, researchers should normalize GPR162 signals to appropriate loading controls (β-Actin has been successfully used in related GPCR studies) and employ statistical methods suitable for the experimental design (e.g., Student's t-test for two-group comparisons).

How should researchers approach cross-species reactivity testing for GPR162 antibodies?

Testing cross-species reactivity of GPR162 antibodies requires a methodical approach to ensure reliable results across different species samples:

• Sequence analysis prerequisites:

  • Compare GPR162 sequences across target species to identify conserved regions

  • Determine the antibody's epitope location and its conservation across species

  • Predict potential cross-reactivity based on epitope homology percentages

• Empirical validation methodology:

  • Test antibodies on positive control samples from each species of interest

  • Use multiple detection techniques (WB, IHC, ELISA) as reactivity may vary by application

  • Include appropriate negative controls (knockdown or knockout samples when available)

• Application-specific considerations:

  • For Western blotting: Be aware that GPR162 may migrate differently in different species

  • For IHC: Optimize fixation protocols for each species' tissue characteristics

  • For ELISA: Validate detection limits for each species separately

What controls are essential for validating GPR162 antibody specificity?

Validating GPR162 antibody specificity requires implementing multiple control strategies to ensure experimental rigor:

• Positive controls:

  • Overexpression systems using flag-tagged GPR162 constructs allow parallel detection with anti-flag and anti-GPR162 antibodies

  • Tissues or cell lines with known high GPR162 expression verified by orthogonal methods

  • Recombinant GPR162 protein standards for calibration in quantitative assays

• Negative controls:

  • siRNA or shRNA knockdown samples (using validated sequences such as 5'-GGCACCTGTGACGACTAC-3')

  • CRISPR/Cas9 knockout cells or tissues when available

  • Isotype controls matched to the primary antibody host species and class

• Peptide competition assays:

  • Pre-incubation of the antibody with the immunizing peptide should abolish specific signal

  • Titration of blocking peptide can demonstrate specificity quantitatively

  • Non-related peptides should not affect antibody binding

• Cross-validation strategies:

  • Use multiple antibodies targeting different epitopes of GPR162

  • Correlate protein detection with mRNA expression using PCR with validated primers

  • Compare results across different experimental techniques (e.g., IHC, WB, IF)

Implementation of these controls helps distinguish true GPR162 signal from non-specific background and artifacts, ensuring reliable and reproducible research findings. Documentation of all validation steps is essential for publication quality research on this target.

How does GPR162 expression correlate with ERK signaling activation?

Interpreting the relationship between GPR162 expression and ERK signaling requires careful analysis of phosphorylation patterns and their statistical significance:

• Experimental approaches for correlation analysis:

  • Western blotting with parallel detection of GPR162, total ERK, and phosphorylated ERK (p-ERK)

  • Quantification of band intensities using densitometry software

  • Normalization of p-ERK to total ERK levels to account for expression variations

• Statistical analysis methods:

  • For overexpression studies, Dunnett's test has been effectively applied when comparing multiple groups to a control

  • Student's t-test is appropriate for direct comparisons between two conditions

  • Data from multiple independent experiments (n≥5) provides robust statistical power

• Interpretation considerations:

  • Research with related GPCRs shows significant increases in ERK phosphorylation upon overexpression

  • Similar methodologies can be applied to GPR162 studies

  • Temporal dynamics should be considered, as ERK phosphorylation may be transient

Available data from related GPCR studies indicates that overexpression in both HEK-293T and N2A cells leads to statistically significant increases in ERK phosphorylation compared to control groups (P<0.05 or P<0.01) . While specific GPR162 data is limited in the provided information, these methodological approaches provide a framework for investigating GPR162's potential role in ERK signaling. The data should be presented with clear statistical parameters and appropriate error bars representing standard error of the mean.

What approaches can differentiate GPR162 from other closely related GPCRs?

Differentiating GPR162 from other structurally similar GPCRs requires integrated methodological strategies:

• Molecular identification approaches:

  • PCR-based discrimination using highly specific primers for GPR162 (forward: 5'-CTCGTCGGGAGTGCGTCT-3', reverse: 5'-GTGGGTGTCTTGGTGCACAG-3')

  • Restriction fragment length polymorphism (RFLP) analysis of PCR products

  • Sequencing validation of amplified products

• Protein-level discrimination:

  • Epitope mapping to identify unique regions in GPR162

  • Western blotting under high-stringency conditions to minimize cross-reactivity

  • 2D gel electrophoresis for separation based on both molecular weight and isoelectric point

• Functional differentiation strategies:

  • Selective knockdown using validated shRNA sequences specific to GPR162

  • Complementation studies with selective receptor agonists/antagonists

  • Bioinformatic analysis of signaling pathways unique to GPR162

• Expression pattern analysis:

  • Comparative tissue distribution studies

  • Co-expression analysis with known interaction partners

  • Subcellular localization patterns through fractionation and imaging

By implementing multiple differentiation approaches, researchers can establish GPR162's unique identity among the GPCR family. This multi-faceted approach is particularly important for orphan GPCRs where functional characterization may be incomplete and structural similarities could confound single-method identification approaches.

How can researchers analyze post-translational modifications of GPR162?

Analysis of GPR162 post-translational modifications (PTMs) requires specialized techniques to detect and characterize these important regulatory features:

• Identification of potential PTMs:

  • The observation of multiple molecular weights (33 kDa and 64 kDa) suggests possible glycosylation, phosphorylation, or other modifications

  • Bioinformatic prediction tools can identify potential modification sites based on sequence analysis

  • Comparison with known PTM patterns in other GPCRs provides additional targets for investigation

• Experimental detection methods:

  • Phosphorylation analysis:

    • Immunoprecipitation with GPR162 antibodies followed by phospho-specific antibody detection

    • Phosphatase treatment to confirm phosphorylation status

    • 32P-orthophosphate metabolic labeling for direct phosphorylation detection

  • Glycosylation analysis:

    • Treatment with glycosidases (PNGase F, Endo H) and observation of mobility shifts

    • Lectin binding assays to characterize glycan structures

    • Mass spectrometry to identify specific glycan compositions

• Functional impact assessment:

  • Site-directed mutagenesis of putative modification sites

  • Correlation of modification status with receptor localization and signaling capacity

  • Temporal analysis of modifications in response to cellular stimuli

• Analysis workflow for mass spectrometry:

  • Immunoprecipitation of GPR162 from cell or tissue lysates

  • In-gel or in-solution digestion with multiple proteases for comprehensive coverage

  • LC-MS/MS analysis with neutral loss scanning for phosphorylation or precursor ion scanning for glycosylation

  • Database searching with variable modification parameters

The different observed molecular weights of GPR162 make it an interesting candidate for PTM studies, as these modifications likely impact receptor function, localization, and signaling capabilities.