GPR107 Antibody, HRP conjugated

What is GPR107 and why is it a significant research target?

GPR107 (G protein-coupled receptor 107), also known as LUSTR1 or KIAA1624, is a multi-pass membrane protein belonging to the LU7TM family. It has emerged as a significant research target due to its biological functions in several key areas:

• It is involved in Golgi-to-ER retrograde transport mechanisms

• It functions as a host factor required for infection by Pseudomonas aeruginosa exotoxin A and Campylobacter jejuni CDT toxins

• It is a promising candidate receptor for neuronostatin, potentially playing an important role in the central control of cardiovascular function

• Its involvement in signal transduction and cell growth regulation makes it relevant for research in cellular biology, neuroscience, and metabolism

The protein's multiple cellular functions and potential implications in disease pathways make it an attractive subject for research aiming to develop novel therapeutic strategies.

What are the key differences between unconjugated and HRP-conjugated GPR107 antibodies?

While standard unconjugated GPR107 antibodies require a secondary detection method, HRP-conjugated versions offer direct detection capabilities:

For standard protocols, HRP-conjugated antibodies streamline experiments but sacrifice the signal amplification that multi-step detection provides. When working with GPR107 specifically, both formats have demonstrated efficacy in detecting the 60-67 kDa protein in various experimental contexts .

What species reactivity is available for GPR107 antibodies?

Available GPR107 antibodies demonstrate reactivity with various species, allowing for cross-species research applications:

When selecting an appropriate antibody for your research, consider both confirmed and predicted reactivity profiles. The bs-16284R antibody offers the broadest predicted cross-reactivity profile, making it potentially valuable for comparative studies across multiple model organisms . Validation in your specific experimental system is always recommended regardless of reported reactivity.

How can I optimize Western blot protocols specifically for GPR107 detection using HRP-conjugated antibodies?

Optimizing Western blot protocols for GPR107 detection requires attention to several key parameters:

Protein Extraction Considerations:

• GPR107 is a membrane protein (60-67 kDa observed weight), requiring effective membrane protein extraction methods

• Include protease inhibitors in lysis buffers to prevent degradation

• For tissues with high lipid content (brain samples), consider specialized extraction buffers

Recommended Protocol Modifications:

• Protein loading: 20-40 μg per lane for cell lysates, 50-70 μg for tissue samples

• Transfer conditions: 100V for 90 minutes using PVDF membrane (preferred over nitrocellulose for membrane proteins)

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody dilution:

  • For unconjugated: 1:500-1:2000 range

  • For HRP-conjugated: Begin with 1:1000, then optimize based on signal-to-noise ratio

• Washing: Extended washing (5 × 5 minutes) to minimize background with direct HRP detection

• Detection: Use ECL substrate with moderate sensitivity; avoid high-sensitivity substrates initially to prevent overexposure

Troubleshooting persistent high background often involves further diluting the HRP-conjugated antibody and extending wash steps rather than shortening incubation times.

What are the critical validation steps needed when working with GPR107 antibodies for novel tissue or cellular applications?

When validating GPR107 antibodies for novel applications, implement a multi-step validation approach:

Essential Validation Steps:

• Positive control selection: Use tissues/cells with known GPR107 expression (cerebellum, HEK-293 cells)

• Knockout/knockdown validation: Compare antibody signal between wild-type and GPR107 KO/KD samples

• Cross-reactivity assessment: Test against closely related family members

• Multi-antibody approach: Use at least two antibodies targeting different epitopes:

  • 25076-1-AP (targets fusion protein Ag18649)

  • PACO00862 (targets internal region of human GPR107)

  • bs-16284R (targets region 301-400/600)

• Orthogonal methods: Confirm protein expression using transcriptomic data

For HRP-conjugated versions specifically:

• Include enzyme-only controls to differentiate between specific binding and intrinsic peroxidase activity

• Perform parallel experiments with unconjugated versions and appropriate secondary antibodies to verify specificity of HRP-conjugated antibody

Creating a validation matrix with multiple antibodies, detection methods, and sample types provides the most robust evidence for antibody specificity in novel applications.

How does GPR107 antibody performance differ across neurological tissue samples compared to peripheral tissues?

GPR107 expression and antibody performance show notable differences between neurological and peripheral tissues:

For neurological tissues specifically, GPR107 antibodies have been validated in mouse cerebellum tissue by Western blot . When working with brain sections for immunohistochemistry or immunofluorescence, antigen retrieval methods significantly impact detection efficiency. Heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) typically yields optimal results for GPR107 detection in fixed brain tissues.

The association between GPR107 and neuronostatin in cardiovascular function regulation suggests potential applications in hypothalamic and brainstem tissues where these regulatory systems are prominent .

What are the recommended protocols for multiplex immunofluorescence including GPR107 detection?

Successful multiplex immunofluorescence incorporating GPR107 detection requires careful consideration of antibody compatibility and detection systems:

Recommended Multiplex Protocol:

• Fixation: 4% PFA for 15 minutes at room temperature (cultured cells) or 24 hours (tissue sections)

• Antigen retrieval: For tissues - heat-mediated retrieval with sodium citrate buffer (pH 6.0)

• Blocking: 10% normal serum (species different from all primary antibodies) with 0.3% Triton X-100

• Primary antibody incubation:

  • GPR107 antibody dilution: 1:50-1:200 for IF applications

  • Co-incubate with other primary antibodies if from different host species

  • Sequential incubation if antibodies are from the same species

• Secondary antibody selection: For GPR107, if not using HRP-conjugated formats, select fluorophores with minimal spectral overlap

• Nuclear counterstaining: DAPI (blue) works well with most multiplex panels

• Mounting: Use anti-fade mounting medium to preserve fluorescence

Compatible Markers for Co-localization Studies:

• Golgi markers (GM130, TGN46) - critical for confirming GPR107's Golgi localization

• ER markers (Calnexin, KDEL) - useful for studying GPR107's role in Golgi-to-ER transport

• Membrane markers (Na+/K+-ATPase) - to confirm cell surface expression

For HRP-conjugated GPR107 antibodies in fluorescent applications, tyramide signal amplification (TSA) systems allow conversion of HRP activity to fluorescent signal while enabling antibody stripping for sequential staining with same-species antibodies.

How can I quantitatively assess GPR107 expression levels across different experimental conditions?

Quantitative assessment of GPR107 expression requires standardized approaches across different detection methods:

Western Blot Quantification:

• Use consistent loading controls (β-actin, GAPDH, or preferably Na+/K+-ATPase for membrane proteins)

• Implement a standard curve with recombinant GPR107 protein

• Use digital image analysis software with background subtraction

• Calculate relative expression as GPR107/loading control ratio

Immunofluorescence Quantification:

• Maintain identical acquisition parameters (exposure time, gain, offset)

• Perform z-stack imaging to capture total cellular expression

• Analyze using automated thresholding algorithms to determine:

  • Mean fluorescence intensity

  • Area of expression

  • Co-localization coefficients with organelle markers

Flow Cytometry Applications:
When using HRP-conjugated GPR107 antibodies for flow cytometry:

• Convert HRP activity to fluorescence using tyramide amplification systems

• Calculate median fluorescence intensity (MFI)

• Present data as fold change relative to control populations

For all quantitative applications, biological and technical replicates (minimum n=3) are essential for statistical validity. When comparing expression across tissues or conditions, normalization to housekeeping proteins is critical, with membrane protein controls being particularly important for GPR107 quantification.

What troubleshooting approaches are recommended for inconsistent GPR107 antibody performance?

When encountering inconsistent results with GPR107 antibodies, systematically address potential issues:

For HRP-conjugated antibodies specifically, enzyme inactivation is a common issue. If signal diminishes over time, consider:

• Avoiding sodium azide in any buffers (inactivates HRP)

• Storing working dilutions at 4°C short-term rather than freezing

• Testing whether reduced HRP activity or antibody binding is the primary issue by comparing with unconjugated versions

If inconsistency persists, switching to a different antibody targeting a different epitope of GPR107 can help determine whether the issue is epitope-specific or sample-related.

How is GPR107 antibody detection being utilized in current neuroscience research?

GPR107 antibody detection has become increasingly valuable in neuroscience research, particularly for investigating:

• Neuronostatin signaling pathways: GPR107 is a promising candidate receptor for neuronostatin, potentially playing an important role in central cardiovascular function regulation

• Neuronal trafficking mechanisms: Given GPR107's role in Golgi-to-ER retrograde transport, research is examining its contribution to protein trafficking in neurons, which is critical for proper synaptic function

• Neuroinflammatory responses: Studies are exploring GPR107's potential interactions with neuroinflammatory pathways, particularly in microglial cells

Current methodological approaches include:

• Co-immunoprecipitation coupled with mass spectrometry to identify GPR107 interaction partners in neuronal cells

• Time-lapse confocal microscopy using fluorescently-tagged GPR107 antibodies to track dynamic trafficking in live neurons

• Proximity ligation assays to validate protein-protein interactions in fixed brain tissue sections

Researchers are encouraged to consider dual-labeling approaches that combine GPR107 detection with markers for specific neuronal populations to better characterize cell type-specific expression patterns across brain regions.

What are the implications of GPR107's role in bacterial toxin interactions for infectious disease research?

GPR107's identified role as a host factor required for infection by bacterial toxins has significant implications for infectious disease research:

Key Research Applications:

• Toxin entry mechanism studies: GPR107 antibodies are being used to block and track interactions with Pseudomonas aeruginosa exotoxin A and Campylobacter jejuni CDT toxins

• Therapeutic target development: Blocking GPR107 using antibodies or small molecules may potentially inhibit toxin entry, representing a host-directed therapeutic approach

• Infection susceptibility profiling: Quantifying GPR107 expression levels across tissues may help predict relative susceptibility to toxin-mediated pathology

Experimental Approaches:

• In vitro toxin challenge assays comparing wild-type and GPR107-depleted cells

• Competitive binding studies between toxins and GPR107 antibodies

• Animal models examining correlation between GPR107 expression and toxin sensitivity

When designing experiments to investigate toxin-GPR107 interactions, researchers should consider:

• Cell-specific expression patterns that may explain tissue tropism of toxins

• Potential conformational changes in GPR107 following toxin binding that might affect antibody recognition

• The possibility of developing blocking antibodies that specifically target toxin-binding domains

How can researchers effectively integrate GPR107 antibody data with transcriptomic and proteomic datasets?

Integrating GPR107 antibody-derived data with -omics approaches provides a more comprehensive understanding of this protein's function:

Integration Strategies:

• Correlation analysis: Compare protein expression levels detected by GPR107 antibodies with mRNA expression from RNA-seq or microarray data

  • Calculate Pearson/Spearman correlation coefficients

  • Identify discordant samples for potential post-transcriptional regulation

• Multi-omics visualization:

  • Create heatmaps displaying GPR107 protein expression alongside transcriptomic data

  • Use dimensionality reduction techniques (PCA, t-SNE) to visualize relationships

• Network analysis:

  • Incorporate GPR107 antibody-derived protein interaction data with transcriptomic networks

  • Identify hub genes/proteins that may regulate GPR107 expression or function

Practical Implementation:

• Use standardized sample processing for both antibody-based detection and -omics analyses

• Include spike-in controls for absolute quantification across platforms

• Develop computational pipelines that normalize and integrate data from multiple sources

For researchers new to multi-omics approaches, begin by establishing baseline GPR107 expression across experimental conditions using antibody-based methods, then expand to targeted transcriptomic analysis of genes within the same pathway before scaling to whole-transcriptome or proteome studies.

What quality control measures should be implemented when working with GPR107 antibodies?

Implementing rigorous quality control measures ensures reliable results when working with GPR107 antibodies:

Essential Quality Control Procedures:

• Antibody validation documentation: Maintain records of all validation experiments including positive/negative controls

• Lot-to-lot verification: Test each new antibody lot against a reference lot using standardized samples

• Regular performance monitoring: Include consistent positive controls in each experiment

• Application-specific controls:

  • For WB: Include molecular weight markers and loading controls

  • For IF/IHC: Include secondary-only controls and known positive tissue sections

  • For IP: Include IgG controls and input samples

For HRP-conjugated antibodies specifically:

• Test enzymatic activity periodically using TMB or other HRP substrates

• Monitor signal-to-noise ratio over time to detect potential HRP degradation

• Store according to manufacturer recommendations (typically 4°C, with minimal freeze-thaw cycles)

Establishing standardized protocols with detailed quality control checkpoints enables reliable longitudinal studies and cross-laboratory reproducibility when working with GPR107 antibodies.

How should researchers document and report GPR107 antibody usage in publications?

Proper documentation of GPR107 antibody usage in publications enhances reproducibility and scientific rigor:

Essential Reporting Elements:

• Complete antibody identification:

  • Manufacturer and catalog number (e.g., 25076-1-AP, PACO00862, bs-16284R)

  • Clone designation (if monoclonal)

  • Host species and isotype

  • RRID (Research Resource Identifier) when available (e.g., AB_2879886)

• Validation methodology:

  • Reference to validation studies or include validation data

  • Description of controls used (positive, negative, knockdown)

• Detailed protocols:

  • Sample preparation methods

  • Antibody dilutions and incubation conditions

  • Detection systems and image acquisition parameters

• Quantification methods:

  • Software used for analysis

  • Parameters measured

  • Statistical approaches

Following these documentation practices not only improves publication quality but also contributes to the broader scientific community's understanding of GPR107 biology and antibody performance characteristics across different experimental systems.