GPR152 Antibody

What is the GPR152 protein and what are its key characteristics?

GPR152 (G protein-coupled receptor 152) is a 511 amino acid multi-pass membrane protein belonging to the G-protein coupled receptor 1 family. It is encoded by a gene located on human chromosome 11q13.2 and mouse chromosome 19A . With a molecular weight of approximately 51 kDa, GPR152 functions as an orphan receptor . The protein mediates its activity through G proteins, resulting in adenylyl cyclase activation and elevated intracellular cAMP levels . GPR152 has been associated with diffuse-type gastric cancer, with rs1790761 in GPR152 showing significant allelic associations with disease susceptibility .

What applications are GPR152 antibodies validated for?

Commercial GPR152 antibodies have been validated for multiple applications:

When selecting an antibody for your research, verify the validation data for your specific application, as performance can vary significantly between products and applications .

What are the optimal storage conditions for GPR152 antibodies?

For long-term storage, GPR152 antibodies should be stored at -20°C for up to one year . To avoid degradation, aliquot the antibody to minimize freeze-thaw cycles . For frequent use over short periods (up to one month), storage at 4°C is acceptable . Most commercial GPR152 antibodies are supplied in a buffer containing PBS, sodium azide, and glycerol, which helps maintain stability during storage .

What controls should be included when working with GPR152 antibodies?

When designing experiments with GPR152 antibodies, include the following controls:

• Positive control: Use tissue or cell lysates known to express GPR152 (based on validation data provided by manufacturers)

• Negative control: Include samples from GPR152 knockout models or cell lines with confirmed low/no expression

• Peptide blocking control: Pre-incubate the antibody with immunizing peptide to confirm specificity

• Isotype control: Use non-specific rabbit IgG at the same concentration as the primary antibody

• Secondary antibody-only control: Omit primary antibody to assess non-specific binding of secondary antibody

How can researchers optimize Western blot protocols for GPR152 detection?

Optimizing Western blot protocols for GPR152 detection requires careful consideration of several factors:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors for membrane protein extraction

  • Heat samples at 37°C instead of boiling to prevent aggregation of the multi-pass membrane protein

  • Include 1-2% SDS in loading buffer to ensure complete denaturation

• Gel selection and transfer:

  • Use 10-12% polyacrylamide gels for optimal separation around the 51 kDa range

  • Transfer to PVDF membranes (rather than nitrocellulose) for better retention of hydrophobic proteins

  • Consider semi-dry transfer at lower voltage for longer duration to improve transfer efficiency

• Antibody incubation:

  • For human samples, use recommended dilutions of 1:500-1:1000

  • Extend primary antibody incubation to overnight at 4°C

  • Use 5% BSA instead of milk for blocking and antibody dilution to reduce background

• Detection optimization:

  • Consider using enhanced chemiluminescence systems with high sensitivity

  • Optimize exposure times based on signal intensity

  • For weak signals, consider using HRP-conjugated polymer detection systems

What strategies can improve immunohistochemical detection of GPR152 in tissue samples?

For optimal immunohistochemical detection of GPR152:

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval (HIER) methods using citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0)

  • For formalin-fixed paraffin-embedded (FFPE) tissues, extend HIER time to 20-30 minutes

• Antibody selection and dilution:

  • Use HPA035078 antibody at 1:500-1:1000 dilution as validated for IHC applications

  • Consider testing multiple GPR152 antibodies targeting different epitopes to confirm staining patterns

• Signal amplification:

  • For low-abundance expression, employ tyramide signal amplification (TSA) systems

  • Use polymer-based detection systems rather than standard ABC methods for increased sensitivity

• Background reduction:

  • Include an avidin-biotin blocking step if using biotin-based detection systems

  • Pre-absorb antibodies with tissue lysates from non-expressing tissues

  • Include 0.1-0.3% Triton X-100 in antibody diluent to improve penetration

• Validation approaches:

  • Compare staining patterns with RNA expression data from databases

  • Use tissue microarrays to systematically evaluate staining across multiple tissue types

What considerations are important when using GPR152 antibodies for co-localization studies?

When designing co-localization experiments with GPR152 antibodies:

• Antibody compatibility:

  • Select primary antibodies raised in different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies, consider sequential immunostaining with thorough blocking between rounds

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • For multi-color imaging, select fluorophores with appropriate brightness to compensate for different expression levels

• Sample preparation:

  • For membrane proteins like GPR152, mild permeabilization protocols are preferred (0.1% saponin rather than stronger detergents)

  • Consider using non-aldehyde fixatives like methanol for better preservation of membrane protein epitopes

• Imaging parameters:

  • Use confocal microscopy with appropriate controls for bleed-through

  • Employ spectral unmixing for closely overlapping fluorophores

  • Consider super-resolution techniques for detailed membrane localization studies

• Quantification approaches:

  • Use Pearson's or Mander's coefficients for quantitative co-localization analysis

  • Include appropriate positive and negative co-localization controls

How can GPR152 antibodies be validated for specificity in experimental systems?

Thorough validation of GPR152 antibodies should include:

• Genetic approaches:

  • Test antibody in GPR152 knockout or knockdown models

  • Compare staining/band patterns in cells with confirmed high versus low expression

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide to block specific binding

  • Use gradual concentrations of blocking peptide to demonstrate dose-dependent inhibition

• Orthogonal techniques:

  • Compare protein detection with mRNA expression data

  • Confirm subcellular localization using GPR152-GFP fusion proteins

• Cross-reactivity assessment:

  • Test reactivity against related GPCRs, particularly those with sequence homology

  • Perform immunoprecipitation followed by mass spectrometry to identify all proteins bound by the antibody

• Application-specific validation:

  • For each application (WB, ICC, IHC), perform separate validation experiments

  • Document optimal conditions for each application with appropriate positive and negative controls

What methodological approaches can be used to study GPR152 signaling pathways using antibodies?

To investigate GPR152 signaling pathways:

• Phosphorylation studies:

  • Use phospho-specific antibodies against downstream effectors (e.g., PKA substrates, CREB) to monitor cAMP-dependent signaling

  • Perform time-course experiments after receptor stimulation or inhibition

• Protein-protein interaction analysis:

  • Use GPR152 antibodies for co-immunoprecipitation followed by mass spectrometry

  • Perform proximity ligation assays (PLA) to detect interactions with suspected binding partners in situ

• Trafficking and internalization studies:

  • Use surface biotinylation followed by GPR152 immunoprecipitation to monitor surface expression

  • Perform antibody feeding assays to track internalization in live cells

• Dimerization and complex formation:

  • Use non-denaturing PAGE followed by Western blotting to detect receptor complexes

  • Perform crosslinking experiments prior to immunoprecipitation to stabilize transient interactions

• Spatial organization analysis:

  • Use super-resolution microscopy with GPR152 antibodies to map receptor distribution in membrane microdomains

  • Combine with lipid raft markers to evaluate compartmentalization

How can researchers address common problems with GPR152 antibody-based Western blots?

What are the best approaches for detecting low-abundance GPR152 expression?

For detecting low-abundance GPR152:

• Sample enrichment strategies:

  • Isolate membrane fractions using ultracentrifugation

  • Perform immunoprecipitation before Western blot analysis

  • Use cell lines with highest endogenous expression based on protein atlas data

• Signal amplification methods:

  • For IHC/ICC: Use tyramide signal amplification (TSA)

  • For Western blot: Use high-sensitivity ECL substrates or infrared detection systems

  • For flow cytometry: Consider using fluorescent-labeled tertiary antibodies

• Improved detection systems:

  • Use highly sensitive digital imaging systems with extended exposure capabilities

  • For fluorescence applications, use high-numerical aperture objectives and sensitive cameras

  • Consider single-molecule detection techniques for extremely low abundance

• Protocol modifications:

  • Extend primary antibody incubation times (overnight at 4°C)

  • Increase antibody concentration while balancing specificity

  • Reduce washing stringency slightly to preserve weak signals

How can researchers compare data from different GPR152 antibody sources?

When comparing data generated using different GPR152 antibodies:

• Epitope mapping:

  • Identify the specific regions targeted by each antibody

  • Compare results from antibodies targeting different domains of GPR152

• Cross-validation approaches:

  • Perform side-by-side comparisons using standardized protocols

  • Use identical positive and negative control samples with each antibody

  • Validate findings with orthogonal techniques (e.g., mass spectrometry, RNA-seq)

• Quantitative comparisons:

  • Use titration experiments to determine the linear range for each antibody

  • Normalize signals to appropriate loading controls

  • Consider statistical approaches for comparing results across antibodies

• Documentation standards:

  • Maintain detailed records of antibody catalog numbers, lot numbers, and dilutions

  • Document specific protocol modifications required for each antibody

  • Report complete methodology in publications to facilitate reproduction

How can GPR152 antibodies be used for high-throughput screening applications?

For adapting GPR152 antibody-based assays to high-throughput formats:

• Assay miniaturization:

  • Adapt Western blot protocols to capillary-based systems

  • Develop ELISA or AlphaLISA formats for quantitative GPR152 detection

  • Optimize antibody concentrations for 384 or 1536-well plate formats

• Automated immunocytochemistry:

  • Establish fixed-cell imaging protocols compatible with high-content screening platforms

  • Develop multiplexed staining protocols to evaluate GPR152 alongside pathway markers

  • Create analysis pipelines for quantitative evaluation of subcellular distribution

• Flow cytometry applications:

  • Optimize staining protocols for suspension cells and dissociated tissues

  • Develop multiplexed panels with GPR152 and relevant pathway markers

  • Establish sorting protocols based on GPR152 expression levels

• Validation considerations:

  • Include appropriate controls on each plate/run

  • Develop robust Z' factor calculations to ensure assay quality

  • Create standard curves with recombinant GPR152 or control cell lines

What considerations are important when using GPR152 antibodies for studying heterogeneity in tissue samples?

When investigating GPR152 expression heterogeneity in tissues:

• Sample preparation:

  • Optimize fixation protocols to preserve both morphology and epitope integrity

  • Consider using tissue clearing techniques for thick section imaging

  • Use antigen retrieval methods optimized for GPR152 detection

• Multiplexed detection approaches:

  • Develop sequential immunostaining protocols for multiple markers

  • Consider cyclic immunofluorescence methods for highly multiplexed imaging

  • Combine with RNA in situ hybridization for correlation with transcript levels

• Spatial analysis methods:

  • Apply digital pathology approaches for quantitative spatial analysis

  • Develop algorithms for cell-type identification and neighborhood analysis

  • Use statistical methods to assess spatial correlation with other markers

• Single-cell approaches:

  • Combine with single-cell isolation techniques for subsequent molecular analysis

  • Consider imaging mass cytometry for highly multiplexed protein detection

  • Correlate protein expression with single-cell transcriptomics data

How can researchers effectively incorporate GPR152 antibodies in live-cell imaging studies?

For live-cell imaging applications with GPR152:

• Antibody fragment generation:

  • Develop Fab fragments from GPR152 antibodies for improved penetration

  • Consider single-chain variable fragments (scFvs) for reduced size

  • Use non-perturbative labeling approaches to maintain receptor function

• Labeling strategies:

  • Direct conjugation with small, bright fluorophores (Alexa Fluor, Atto dyes)

  • Use antibody binding fragments conjugated to quantum dots for prolonged imaging

  • Consider photoactivatable or photoswitchable fluorophores for pulse-chase experiments

• Delivery methods:

  • Optimize antibody loading techniques (electroporation, microinjection)

  • Develop cell-penetrating peptide conjugates for non-disruptive delivery

  • Consider expression of intrabodies for intracellular targeting

• Imaging considerations:

  • Minimize laser power and exposure to reduce phototoxicity

  • Use environmental chambers to maintain physiological conditions

  • Employ spinning disk or light sheet microscopy for reduced photodamage