GPR179 Antibody, HRP conjugated

What is GPR179 and why is it important in vision research?

GPR179 (G Protein-Coupled Receptor 179) is an orphan receptor required for signal transduction through retinal depolarizing bipolar cells. It localizes at the dendritic tips of bipolar cells in human retina and plays an essential role in normal visual function . Mutations in GPR179 lead to autosomal recessive complete congenital stationary night blindness (cCSNB), characterized by ON-bipolar retinal cell dysfunction . This makes GPR179 particularly significant for understanding visual pathways.

The receptor forms part of the post-synaptic signaling complex in ON-bipolar neurons. Research has demonstrated that GPR179 interacts with heparan sulfate proteoglycans (HSPGs), including Pikachurin, which is released by photoreceptors . Through proximity ligation assays, researchers have confirmed that the Pikachurin-GPR179 complex forms at characteristic synaptic puncta confined to the outer plexiform layer (OPL), predominantly decorating dendritic tips of PKCα-positive rod ON-bipolar cells . These interactions are crucial for proper synaptic function in the visual pathway.

The study of GPR179 provides valuable insights into visual signal transduction mechanisms and potential therapeutic targets for retinal disorders, making it an important focus in ophthalmology and neuroscience research.

What applications can GPR179 Antibody, HRP conjugated be used for?

GPR179 Antibody, HRP conjugated is designed for multiple research applications, with primary utility in detection and quantification methods. According to available data, this antibody has been validated for several key applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): The most common application, with recommended dilutions ranging from 1:2000 to 1:10000 . The HRP conjugation enables direct detection without secondary antibodies, streamlining protocols.

• Western Blotting (WB): Effective for detecting GPR179 protein in tissue or cell lysates, typically at dilutions between 1:1000-1:5000 . The direct HRP conjugation eliminates secondary antibody incubation steps.

• Dot Blot Analysis: Useful for rapid screening of samples for GPR179 presence before more detailed analyses .

While not the primary applications, some researchers have adapted HRP-conjugated antibodies for immunohistochemistry and immunofluorescence with appropriate optimization. For these applications, more dilute preparations (typically 1:50-1:200) are recommended as starting points .

The direct HRP conjugation provides significant advantages by reducing protocol time, minimizing cross-reactivity issues, and potentially improving signal-to-noise ratios in detection systems.

How does the structure of GPR179 affect antibody selection for retinal research?

GPR179 possesses a complex multi-domain structure that directly impacts antibody selection for retinal research applications. Understanding this structure is crucial for targeting specific functional domains and interpreting experimental results.

GPR179 is characterized by:

• A large N-terminal extracellular domain that interacts with Pikachurin and other heparan sulfate proteoglycans (HSPGs)

• Seven transmembrane domains typical of G protein-coupled receptors

• An intracellular C-terminal domain involved in downstream signaling

When selecting a GPR179 antibody, researchers should consider which domain is recognized by the antibody. For instance, antibodies targeting the extracellular domain (such as those recognizing amino acids 1440-1671) are particularly valuable for studying protein-protein interactions at the synapse . These antibodies can help visualize the localization of GPR179 at the dendritic tips of bipolar cells and its interactions with presynaptic proteins released from photoreceptors.

Research has demonstrated that protein localization studies using appropriate anti-GPR179 antibodies show characteristic punctate staining patterns in the outer plexiform layer, corresponding to the dendritic tips of bipolar cells . The distinct subcellular localization of GPR179 requires careful antibody selection to ensure proper epitope accessibility in fixed tissue samples.

For mutation studies, antibodies targeting different domains can help determine how specific mutations affect protein expression, trafficking, and localization. For example, research has shown that missense mutations p.Tyr220Cys, p.Gly455Asp, and p.His603Tyr lead to severely reduced cell surface localization of GPR179, while p.Asp126His does not .

What are the recommended protocols for GPR179 detection in retinal tissue?

Detecting GPR179 in retinal tissue requires specific protocols optimized for this challenging tissue type and the protein's unique localization pattern. Based on published methodologies, the following approach is recommended:

Tissue Preparation:

• Fix freshly dissected retinal tissue in 4% paraformaldehyde for 15-30 minutes (overfixation can mask epitopes)

• Cryoprotect in sucrose gradient (10%, 20%, 30%) and embed in OCT compound

• Section at 10-12 μm thickness to preserve retinal architecture while allowing antibody penetration

Immunohistochemistry Protocol:

• Antigen Retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes at 95°C

• Permeabilization: Treat with 0.3% Triton X-100 in PBS for 30 minutes to improve antibody access to dendritic tips

• Blocking: Block with 10% normal serum + 1% BSA + 0.1% Triton X-100 for at least 2 hours at room temperature

• Primary Antibody: Apply GPR179 Antibody, HRP conjugated at 1:50-1:200 dilution in blocking buffer; incubate overnight at 4°C

• Signal Development: For chromogenic detection, develop with DAB substrate; for fluorescent detection, use tyramide signal amplification systems compatible with HRP

Controls and Validation:

• Positive Control: Include normal human retina sections known to express GPR179

• Negative Control: Omit primary antibody or use tissue from GPR179-deficient models

• Specificity Control: Pre-incubate antibody with recombinant GPR179 protein (1440-1671AA immunogen region)

When analyzing results, GPR179 should appear as distinct punctate labeling in the outer plexiform layer, corresponding to dendritic tips of bipolar cells . This characteristic pattern serves as an internal validation of specific staining.

For co-localization studies, combine GPR179 staining with markers of bipolar cells (e.g., PKCα) and photoreceptor synaptic terminals to confirm the correct synaptic localization .

How should GPR179 Antibody, HRP conjugated be stored and handled for optimal performance?

Proper storage and handling of GPR179 Antibody, HRP conjugated is critical for maintaining its activity and specificity. Follow these guidelines to ensure optimal antibody performance:

Storage Conditions:

• Store unopened antibody at -20°C for long-term storage

• For frequent use, store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles by preparing working aliquots upon first thaw

Buffer Information:
The antibody is typically supplied in a storage buffer containing:

• 50% Glycerol

• 0.01M PBS, pH 7.4

• 0.03% Proclin 300 or similar preservative

This formulation helps maintain antibody stability during storage and handling.

Handling Recommendations:

• Centrifuge the antibody vial briefly before opening to collect liquid at the bottom

• Keep the antibody on ice when in use

• Protect from prolonged exposure to light

• Return to appropriate storage temperature immediately after use

• Prepare working dilutions fresh for each experiment

Critical Precautions:

• NEVER use sodium azide in buffers with HRP-conjugated antibodies, as it inhibits HRP activity

• Avoid exposure to oxidizing and reducing agents that can damage the HRP enzyme

• Minimize exposure to extreme pH conditions

Working Solution Preparation:

• Dilute only the amount needed for immediate use

• Use high-quality, filtered buffers for dilution

• For ELISA applications, optimal dilutions range from 1:2000-1:10000

• For Western blot applications, start with 1:1000-1:5000 dilutions

• Store reconstituted antibody at 4°C and use within 24 hours for best results

When properly stored and handled, GPR179 Antibody, HRP conjugated should maintain its activity and specificity, ensuring reliable experimental results and minimizing variability between assays.

How can researchers optimize GPR179 Antibody, HRP conjugated for proximity ligation assays studying GPR179-Pikachurin interactions?

Proximity Ligation Assay (PLA) is a powerful technique for studying protein-protein interactions such as the documented GPR179-Pikachurin complex in retinal tissue. Optimizing GPR179 Antibody, HRP conjugated for PLA requires several specialized approaches:

HRP Deconjugation Strategy:
Since standard PLA protocols require unconjugated primary antibodies, the HRP conjugation presents a challenge that must be addressed through one of two approaches:

• Chemical Quenching Method:

  • Treat the HRP-conjugated antibody with 10 mM sodium azide + 10 mM hydrogen peroxide for 30 minutes at room temperature

  • Dialyze against PBS overnight to remove quenching agents

  • Proceed with oligonucleotide conjugation for PLA

• Alternative Approach:

  • Use an unconjugated anti-GPR179 antibody recognizing a different epitope

  • Pair with antibodies against Pikachurin for PLA

Protocol Optimization for Retinal Tissue:

• Tissue Preparation:

  • Use thin sections (8-10 μm) of properly fixed retinal tissue

  • Perform antigen retrieval with citrate buffer (pH 6.0) for 20 minutes

  • Block extensively (3+ hours) with Duolink blocking solution supplemented with 1% BSA

• Antibody Dilutions:

  • For deconjugated GPR179 antibody: use 1:100-1:200 (more concentrated than in standard immunoassays)

  • For Pikachurin antibody: use 1:100-1:500 depending on antibody quality

• Critical Controls:

  • Negative Controls: Omit one primary antibody; use retinal tissue from GPR179-deficient models

  • Competition Control: Pre-incubate sections with purified ecto-GPR179-Fc to block specific interactions

  • Positive Controls: Include known interacting partners validated in previous studies

Research has demonstrated that properly optimized PLA for GPR179-Pikachurin shows characteristic punctate signals confined to the outer plexiform layer, corresponding to synapses between photoreceptors and bipolar cells . The PLA signals should decorate dendritic tips of PKCα-positive rod ON-bipolar cells but may also be present in PKCα-negative sites, indicating interactions at both rod and cone synapses .

For quantification, count PLA puncta within the OPL and normalize to synapse density markers to enable meaningful comparisons between experimental conditions.

What strategies can resolve technical challenges when detecting large membrane proteins like GPR179 in Western blots?

Detecting large membrane proteins like GPR179 (~250 kDa) by Western blot presents significant technical challenges that require specialized approaches. Here are comprehensive strategies to overcome these difficulties:

Protein Extraction Optimization:

• Membrane Protein Extraction:

  • Use specialized membrane protein extraction buffers containing 1-2% non-ionic detergents (Triton X-100 or NP-40)

  • Include protease inhibitor cocktail to prevent degradation

  • Solubilize at 4°C for 1-2 hours with gentle rotation

  • Avoid boiling samples (heat at 37°C for 30 minutes instead)

• Sample Preparation:

  • Add sample buffer containing 2% SDS and 8M urea to improve solubilization

  • Include reducing agent (DTT or β-mercaptoethanol) at appropriate concentration

  • Avoid freeze-thaw cycles of prepared samples

Electrophoresis Parameters:

• Gel Selection:

  • Use low percentage (4-6%) or gradient (4-15%) gels to resolve high molecular weight proteins

  • Consider commercial pre-cast gels optimized for large proteins

• Running Conditions:

  • Use cold running buffer and run at lower voltage (60-80V) for extended time

  • Add 0.5% SDS to running buffer to maintain protein solubility

  • Run until the 250 kDa marker is well-resolved (may take 2-3 hours)

Transfer Optimization:

• Transfer Method:

  • Use wet transfer method exclusively (semi-dry systems inadequate for large proteins)

  • Transfer overnight at 30V, 4°C

  • Add 0.05% SDS to transfer buffer to maintain protein solubility

• Membrane Selection:

  • PVDF membranes with 0.45 μm pore size are optimal for large proteins

  • Pre-activate PVDF with methanol followed by equilibration in transfer buffer

Detection Protocol:

• Antibody Application:

  • Block membrane extensively (overnight at 4°C) with 5% non-fat dry milk in TBS-T

  • Incubate with GPR179 Antibody, HRP conjugated at 1:1000 dilution in 1% BSA in TBS-T overnight at 4°C

  • Wash extensively (5 x 10 minutes) in TBS-T

• Signal Development:

  • Use enhanced chemiluminescent substrate with extended signal duration

  • Capture multiple exposures (15 seconds, 1 minute, 5 minutes, 15 minutes)

  • For weak signals, consider using signal enhancers compatible with HRP

Validation Controls:

• Positive Control: Include human retinal lysate (known to express GPR179)

• Negative Control: Include tissue known not to express GPR179

• Size Verification: Use high molecular weight protein ladder covering 250+ kDa range

When properly optimized, Western blot for GPR179 should show a specific band at approximately 250 kDa in retinal samples, with minimal non-specific binding or background signal.

How can researchers validate the specificity of GPR179 Antibody, HRP conjugated for their experimental models?

Comprehensive validation of GPR179 Antibody, HRP conjugated specificity is essential before conducting definitive experiments. A multi-layered approach ensures reliable results across different experimental systems:

1. Expression System Validation:

2. Epitope-Specific Validation:

GPR179 has several domains, and knowing which region the antibody recognizes is critical:

• For antibodies raised against the recombinant Human GPR179 protein region (1440-1671AA), expression of this specific domain in heterologous cells should yield positive signals

• Testing the antibody against truncated versions of GPR179 can map the exact epitope recognition site

• Cross-validation with another GPR179 antibody recognizing a different epitope provides additional confirmation

3. Application-Specific Validation:

For ELISA:

• Prepare a standard curve using recombinant GPR179 protein

• Test antibody at multiple dilutions (1:2000, 1:5000, 1:10000)

• Determine limit of detection and dynamic range

• Include negative controls (buffer only, irrelevant protein)

For Western Blot:

• Run gradient gel with positive control, experimental sample, and negative control

• Test multiple dilutions to identify optimal concentration

• Compare results between reducing and non-reducing conditions

• Validate with orthogonal detection method (different GPR179 antibody)

4. Cross-Reactivity Assessment:

If working with non-human models:

• Perform sequence alignment between human GPR179 and your model organism's GPR179

• Focus on the immunogen region (1440-1671AA)

• Test antibody against recombinant GPR179 from your model organism

• Compare staining patterns between human and model organism tissues

Research has shown that GPR179 localizes specifically at dendritic tips of bipolar cells in human retina . In proper validation experiments, this characteristic punctate pattern in the outer plexiform layer should be observed with specific GPR179 antibodies.

For the HRP-conjugated format specifically, additional controls should include testing for potential interference from the conjugation process by comparing with unconjugated versions of the same antibody.

What approaches can enhance detection sensitivity for low abundance GPR179 in diverse retinal samples?

Detecting low abundance GPR179 in retinal samples requires specialized approaches to amplify signal while maintaining specificity. The following methodological enhancements can significantly improve detection sensitivity:

Signal Amplification Techniques:

• Tyramide Signal Amplification (TSA):

  • Apply GPR179 Antibody, HRP conjugated at a 1:2000-1:5000 dilution

  • Use fluorophore-conjugated tyramide (10-15 minutes at room temperature)

  • HRP catalyzes deposition of multiple fluorophore molecules, amplifying signal 10-100 fold

  • This technique is particularly valuable for detecting GPR179 in samples with low expression

• Enhanced Chemiluminescence (ECL) Optimization:

  • For Western blots, use high-sensitivity ECL substrates

  • Extend exposure times (up to 30 minutes) for very low abundance samples

  • Consider using ECL substrates with signal enhancers specifically designed for HRP

• Extended Antibody Incubation:

  • Incubate with GPR179 Antibody, HRP conjugated at 4°C for 48-72 hours

  • Use a slightly higher concentration (1:500-1:1000) than standard protocols

  • Maintain stringent washing to minimize background signal increase

Sample Preparation Enhancements:

• Protein Enrichment Strategies:

  • For Western blot: Load higher protein amounts (50-100 μg/lane)

  • For tissue sections: Use thicker sections (12-16 μm) for immunohistochemistry

  • Consider membrane protein enrichment protocols before analysis

• Antigen Retrieval Optimization:

  • Test multiple antigen retrieval methods:

    • Heat-induced epitope retrieval with citrate buffer (pH 6.0)

    • Tris-EDTA buffer (pH 9.0)

    • Enzymatic retrieval with proteases

  • Optimize retrieval time (15-30 minutes) for maximum epitope exposure while preserving tissue architecture

Detection Protocol Refinements:

• Background Reduction:

  • Extended blocking (3+ hours) with 5% BSA + 3% normal serum

  • Addition of 0.1% Tween-20 to all buffers

  • Include 0.3M NaCl in antibody dilution buffer to reduce non-specific ionic interactions

• Signal-to-Noise Enhancement:

  • Pre-absorb antibody with liver powder (100 mg/ml) to reduce non-specific binding

  • Use low-fluorescence microscope slides and mounting media for fluorescence applications

  • Employ image acquisition settings that maximize signal while minimizing background

Specialized Imaging Techniques:

• Confocal Microscopy:

  • Use high-NA objectives (1.3-1.4) to maximize light collection

  • Employ narrow bandpass filters to reduce autofluorescence

  • Consider spectral unmixing to separate specific signal from tissue autofluorescence

  • Use z-stack acquisition with deconvolution to improve signal-to-noise ratio

• Super-Resolution Microscopy:

  • For detailed localization studies of GPR179 at synaptic structures, consider STED or STORM super-resolution techniques

  • These approaches can resolve GPR179 localization with 20-50 nm precision

These enhancements should be systematically tested and optimized for specific sample types to achieve maximum sensitivity while maintaining specificity in GPR179 detection.

How can researchers troubleshoot non-specific binding issues when using GPR179 Antibody, HRP conjugated?

Non-specific binding is a common challenge when working with antibodies, especially in complex tissues like retina. For GPR179 Antibody, HRP conjugated, systematic troubleshooting approaches can resolve these issues:

1. Systematic Optimization Approach:

2. Application-Specific Optimizations:

For ELISA Applications:

• Antibody Titration Matrix:

  • Test GPR179 Antibody, HRP conjugated at 6 dilutions (1:1000 to 1:20000)

  • Calculate signal-to-noise ratio for each dilution

  • Select optimal dilution with highest signal-to-noise ratio

• Buffer Optimization:

  • Test different diluents: PBS-T, TBS-T, PBS-T + 0.1% BSA, PBS-T + 1% BSA

  • Test different blocking agents: 1-5% BSA, 1-5% casein, commercial blockers

  • Identify combination yielding lowest background with preserved specific signal

For Western Blot Applications:

• Membrane Treatment Protocol:

  • After transfer, rinse membrane in TBS-T for 5 minutes

  • Block with 5% non-fat dry milk in TBS-T for minimum 2 hours at room temperature

  • Wash 3 x 5 minutes in TBS-T

  • Incubate with GPR179 Antibody, HRP conjugated diluted in 1% BSA in TBS-T overnight at 4°C

  • Wash 5 x 5 minutes in TBS-T before developing

For Immunohistochemistry Applications:

• Tissue Preparation Optimization:

  • Test multiple fixation protocols (2%, 4%, 8% PFA; 15 min, 30 min, 1 hour)

  • Compare multiple antigen retrieval methods

  • Determine optimal combination for GPR179 detection

• Advanced Blocking Strategy:

  • Sequential blocking: 30 min with 0.3% H₂O₂, then 2 hours with 5% normal serum + 1% BSA

  • Include additional blockers: 0.1% fish gelatin, 0.1% cold water fish skin gelatin, 0.5% glycine

3. Retina-Specific Considerations:

• Autofluorescence Reduction:

  • Pre-treat sections with 0.1% Sudan Black B in 70% ethanol for 20 minutes

  • Wash thoroughly in PBS before antibody application

• Cross-Adsorption Protocol:

  • Pre-adsorb GPR179 Antibody, HRP conjugated with liver powder (100 mg/ml) for 1 hour

  • Centrifuge at 10,000g for 10 minutes

  • Use supernatant for staining

4. Validation Controls:

• Antibody Specificity Controls:

  • Pre-absorption with recombinant GPR179 protein (immunogen region 1440-1671AA)

  • Comparison with alternative GPR179 antibody recognizing different epitope

  • Testing on GPR179-deficient tissue (if available)

• Technical Controls:

  • Secondary antibody only (for detecting endogenous peroxidase or non-specific binding)

  • Isotype control (irrelevant HRP-conjugated antibody of same isotype)

  • Concentration-matched non-specific HRP-conjugated IgG

Research has shown that specific GPR179 staining should appear as punctate labeling in the outer plexiform layer, corresponding to dendritic tips of bipolar cells . Any staining pattern that deviates significantly from this characteristic distribution should be investigated for potential non-specific binding issues.