GUCY2D Antibody, HRP conjugated

What is GUCY2D and why is it significant in retinal research?

GUCY2D encodes retinal guanylyl cyclase 1 (RetGC1 or ROS-GC1), a critical enzyme in the phototransduction cascade that catalyzes the conversion of GTP to cGMP in photoreceptor cells. This protein is particularly significant because mutations in the GUCY2D gene are associated with Leber congenital amaurosis type 1 (LCA1), accounting for 6-21% of all LCA cases . LCA represents a group of severe congenital retinal diseases characterized by significant visual impairment from birth. The study of GUCY2D provides critical insights into normal retinal physiology and the pathophysiology of inherited retinal degenerations, making antibodies against this protein essential tools for vision research.

What are the key applications for HRP-conjugated GUCY2D antibodies?

HRP-conjugated GUCY2D antibodies serve multiple experimental purposes across different methodologies:

The search results indicate that GUCY2D antibodies targeting different epitopes have been validated for these applications, with many commercially available options being suitable for Western blotting, IHC, and ELISA .

Which regions of GUCY2D protein are commonly targeted by commercial antibodies?

Commercial antibodies target various domains of the GUCY2D protein, each with specific advantages for different experimental applications:

This diversity allows researchers to select antibodies targeting epitopes that are preserved in their experimental conditions or unaffected by known disease mutations. The middle region antibodies (AA 547-578) appear particularly versatile across multiple applications .

How do GUCY2D mutations affect antibody recognition in retinal disease research?

Mutations in GUCY2D can significantly impact antibody binding, creating challenges for immunodetection in disease models. The search results describe three novel mutations in GUCY2D associated with LCA1: c.139delC (p.Ala49Profs*36), c.835G>A (p.Asp279Asn), and c.2783G>A (p.Gly928Glu) . These mutations illustrate different mechanisms that can affect antibody recognition:

• Truncation mutations (like c.139delC) may result in shortened proteins that lack the epitope entirely, particularly for antibodies targeting C-terminal regions.

• Missense mutations (like c.835G>A and c.2783G>A) can alter protein conformation, potentially masking or exposing different epitopes even when the amino acid sequence of the epitope itself remains intact.

• Mutations affecting protein stability may lead to reduced target abundance, requiring more sensitive detection methods.

To address these challenges, researchers should:

• Select antibodies targeting epitopes distant from common mutation sites

• Use multiple antibodies targeting different domains for comprehensive assessment

• Include appropriate controls from unaffected individuals and heterozygous carriers

• Correlate antibody detection with functional assays (such as the HPLC-MS/MS method for cGMP measurement described in the research)

What optimization strategies improve signal-to-noise ratio with HRP-conjugated GUCY2D antibodies?

Optimizing signal-to-noise ratio is critical for generating reliable data with HRP-conjugated GUCY2D antibodies. Advanced researchers should consider:

Blocking Optimization:

• Test multiple blocking agents: 3-5% BSA versus 5% non-fat dry milk in TBS-T

• For tissues with high endogenous biotin, avoid BSA (which may contain biotin traces)

• Include a peroxidase quenching step (0.3% H₂O₂) when working with tissues having high endogenous peroxidase activity

Antibody Dilution Titration:

• Create a systematic dilution series (1:500, 1:1000, 1:2000, 1:5000)

• Evaluate based on both signal intensity and background

• Different applications require different optimal dilutions (typically more concentrated for IHC than for Western blot)

Substrate Selection:

• Enhanced chemiluminescence (ECL) systems offer superior sensitivity for Western blotting

• DAB (3,3'-Diaminobenzidine) provides permanent signals for IHC applications

• TMB (3,3',5,5'-Tetramethylbenzidine) works well for ELISA quantification

Advanced Signal Enhancement:

• Tyramide Signal Amplification (TSA) can amplify HRP signals 10-100 fold for detecting low abundance targets

• Metal-enhanced DAB (using cobalt or nickel) can improve sensitivity in IHC applications

These optimization strategies should be systematically tested and documented for reproducible results across experiments.

How can GUCY2D expression be quantitatively measured using HRP-conjugated antibodies?

Quantitative measurement of GUCY2D expression using HRP-conjugated antibodies can be approached through several methodologies:

Western Blot Densitometry:

• Separate proteins by SDS-PAGE and transfer to PVDF membranes

• Incubate with optimally diluted HRP-conjugated GUCY2D antibody

• Develop using a chemiluminescent substrate with linear dynamic range

• Capture images using a digital imaging system

• Perform densitometric analysis, normalizing GUCY2D band intensity to a housekeeping protein

Quantitative ELISA:
The search results describe a sandwich ELISA approach for GUCY2D quantification :

• Antibody-coated microplates capture GUCY2D from samples

• Detection antibody binds to captured GUCY2D

• HRP-conjugated streptavidin (or direct HRP-conjugated antibody) provides enzymatic signal

• Substrate development produces colorimetric signal proportional to GUCY2D concentration

• Quantification against a standard curve of known GUCY2D concentrations

Image Cytometry for IHC:

• Stain tissue sections using standardized IHC protocols with HRP-conjugated GUCY2D antibody

• Develop with DAB or other chromogenic substrate

• Capture digital images under consistent conditions

• Use image analysis software to quantify staining intensity

• Compare to calibrated standards if absolute quantification is needed

For all quantitative applications, rigorous controls and validation of the linear detection range are essential for reliable results.

What is the recommended protocol for using HRP-conjugated GUCY2D antibodies in Western blot?

The following optimized protocol is recommended for Western blot analysis using HRP-conjugated GUCY2D antibodies:

Sample Preparation:

• Extract proteins from tissues or cells using RIPA buffer containing protease inhibitors

• Determine protein concentration using BCA or Bradford assay

• Prepare samples by mixing with Laemmli buffer (containing SDS and β-mercaptoethanol)

• Heat samples at 95°C for 5 minutes

Gel Electrophoresis and Transfer:

• Load 20-50 μg of protein per lane on an 8% SDS-PAGE gel (GUCY2D is approximately 115-120 kDa)

• Run gel at 100-120V until adequate separation

• Transfer proteins to PVDF membrane at 100V for 1 hour or 30V overnight at 4°C

Immunodetection:

• Block membrane with 5% non-fat dry milk or 3-5% BSA in TBS-T for 1 hour at room temperature

• Dilute HRP-conjugated GUCY2D antibody (typically 1:1000 to 1:5000) in blocking buffer

• Incubate membrane with diluted antibody solution for 2 hours at room temperature or overnight at 4°C

• Wash membrane 4-5 times with TBS-T, 5 minutes each

• Proceed directly to detection (no secondary antibody needed)

Detection:

• Prepare ECL substrate according to manufacturer's instructions

• Apply substrate to membrane and incubate for 1 minute

• Capture chemiluminescent signal using X-ray film or digital imaging system

• For quantitative analysis, ensure exposure is within linear range

Controls:

• Include positive control (retinal tissue extract)

• Include molecular weight markers

• Consider running a loading control (β-actin or GAPDH) for normalization

• For validation, include a peptide competition control

This protocol may require optimization based on the specific HRP-conjugated GUCY2D antibody being used and the nature of the samples being analyzed .

How should GUCY2D antibodies be used for immunohistochemistry of retinal tissues?

Immunohistochemical detection of GUCY2D in retinal tissues requires specific considerations due to the complex architecture and delicate nature of retinal tissue:

Tissue Preparation:

• Fix freshly isolated retinal tissue in 4% paraformaldehyde for 24 hours at 4°C

• Process and embed in paraffin, or prepare frozen sections for better epitope preservation

• Section at 5-7 μm thickness, ensuring proper orientation to visualize retinal layers

Antigen Retrieval (for paraffin sections):

• Deparaffinize and rehydrate sections through xylene and graded alcohols

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Maintain sections at 95-98°C for 15-20 minutes, then cool gradually

Immunostaining Protocol:

• Block endogenous peroxidase activity with 0.3% H₂O₂ for 10 minutes

• Apply protein blocking solution (5% normal serum from same species as secondary antibody would be used)

• Incubate with HRP-conjugated GUCY2D antibody at optimized dilution (typically starting at 1:100)

• Incubate overnight at 4°C in a humidified chamber

• Wash thoroughly with PBS (3 × 5 minutes)

• Develop with DAB substrate for optimal time (2-10 minutes, monitoring microscopically)

• Counterstain with hematoxylin, dehydrate, and mount

Specialized Considerations for Retinal Tissue:

• Include positive control tissue from wild-type retina of the same species

• Consider double-labeling with photoreceptor markers to localize GUCY2D expression

• For human samples, account for postmortem time as it affects immunoreactivity

• When studying disease models, examine both central and peripheral retina

The search results indicate that several GUCY2D antibodies are validated for IHC in paraffin-embedded sections .

What troubleshooting approaches address weak or absent signals with GUCY2D antibodies?

When encountering weak or absent signals with GUCY2D antibodies, a systematic troubleshooting approach can identify and resolve the issue:

Antibody Validation Issues:

• Verify antibody reactivity with positive control samples (retinal tissue known to express GUCY2D)

• Check antibody storage conditions (HRP-conjugated antibodies should be stored at 2-8°C for short-term or -20°C for long-term)

• Test a new lot of antibody if possible

• Consider antibodies targeting different epitopes of GUCY2D

Sample-Related Issues:

• Evaluate protein extraction efficiency for membrane proteins

• Check for protein degradation (use fresh protease inhibitors)

• Verify protein loading amount (consider increasing concentration)

• For fixed tissues, assess fixation conditions (overfixation can mask epitopes)

Protocol Optimization:

• For Western Blot:

  • Increase antibody concentration

  • Extend incubation time (overnight at 4°C)

  • Try alternative membrane types (PVDF may be better than nitrocellulose)

  • Use more sensitive detection substrates

• For IHC:

  • Optimize antigen retrieval conditions (test different buffers and durations)

  • Reduce washing stringency

  • Extend antibody incubation time

  • Try signal amplification systems

• For ELISA:

  • Check coating efficiency

  • Use assay diluents that enhance signal-to-noise ratio

  • Optimize antibody concentration based on standard curve performance

HRP-Specific Considerations:

• Verify substrate freshness and preparation

• Ensure absence of sodium azide in working solutions (inhibits HRP)

• Protect from light during development

• Check for interfering substances in samples

Systematic documentation of troubleshooting steps will help identify the critical variables affecting GUCY2D detection in specific experimental systems.

What are the optimal storage conditions for maintaining HRP-conjugated GUCY2D antibody activity?

Proper storage is critical for maintaining the activity and specificity of HRP-conjugated GUCY2D antibodies. According to the search results, the following conditions are recommended :

Temperature Guidelines:

• Short-term storage (≤1 month): 2-8°C

• Long-term storage: -20°C in small aliquots to minimize freeze-thaw cycles

• Avoid storage at room temperature except during use

• Never store at -80°C as extreme cold can denature antibodies

Buffer Composition:

• Preferred storage buffer: PBS or TBS (pH 7.2-7.6) with:

  • 50% glycerol for frozen storage to prevent ice crystal formation

  • 0.02-0.05% sodium azide as preservative (note: azide can inhibit HRP activity, so use minimal concentrations)

  • 1-2% BSA or other stabilizing protein

Physical Storage Conditions:

• Container material: Dark glass vials or polypropylene tubes (not polystyrene)

• Light protection: Store in dark conditions as light exposure can affect both the antibody and the HRP conjugate

• Position: Store upright to minimize contact with container cap

Working Stock Handling:

• Upon receipt, divide into small single-use aliquots (10-50 μL)

• Use diluted antibody within 24 hours

• Minimize freeze-thaw cycles; each cycle can reduce activity by 10-20%

• Document lot numbers, receipt dates, and performance metrics

Following these storage guidelines will help maintain the activity and specificity of HRP-conjugated GUCY2D antibodies over time, ensuring reliable experimental results.

How does conjugation to HRP affect antibody stability and performance?

HRP conjugation introduces specific considerations that affect antibody stability and performance in research applications:

Stability Considerations:

• HRP-conjugated antibodies typically have shorter shelf-life than unconjugated antibodies

• The enzymatic component (HRP) is more susceptible to denaturation than the antibody portion

• Stability can be compromised by repeated freeze-thaw cycles, extreme pH, or oxidizing agents

Performance Characteristics:

• Direct detection without secondary antibody reduces background but may provide less signal amplification compared to two-step detection systems

• HRP conjugation may slightly reduce antibody binding affinity due to steric hindrance

• Lower activity over time is normal and should be accounted for by periodic revalidation

Storage Buffer Requirements:

• Avoid sodium azide in working solutions as it inhibits HRP activity

• Thimerosal and other mercury-containing preservatives can also inhibit HRP

• Stabilizing proteins (BSA, casein) help maintain conjugate activity

Special Handling Considerations:

• Protect from strong light exposure during storage and use

• Maintain pH between 6.0-8.0 for optimal HRP stability

• Consider specialized stabilizing diluents for working solutions

The protocols provided in search result for ELISA applications with GUCY2D antibodies highlight the importance of proper buffer composition and handling techniques for maintaining optimal performance of HRP-conjugated antibodies.

How can GUCY2D antibodies contribute to the study of retinal diseases?

GUCY2D antibodies serve as critical tools for investigating the molecular mechanisms of retinal diseases, particularly Leber congenital amaurosis type 1 (LCA1). The search results highlight several key research applications:

Disease Mechanism Investigation:

• Examine expression levels and localization of wild-type versus mutant GUCY2D protein in patient samples

• The identified mutations (c.139delC, c.835G>A, c.2783G>A) affect different regions of the protein and likely have distinct impacts on expression and function

• Antibodies targeting different domains can help characterize the effects of specific mutations

Genotype-Phenotype Correlation:

• Compare GUCY2D expression patterns between patients with different mutation types

• Link protein expression levels to disease severity and progression

• Examine heterozygous carriers to understand dosage effects

Therapeutic Development Support:

• Monitor protein expression in gene therapy studies

• Validate animal models of GUCY2D-related diseases

• Screen for compounds that stabilize mutant GUCY2D protein

Functional Studies:

• Couple immunodetection with functional assays of guanylate cyclase activity

• The search results describe using HPLC-MS/MS to measure cGMP production as a functional readout of GUCY2D activity

• Correlate protein levels with enzymatic function in different disease states

Diagnostics Development:

• The search results include an ELISA kit for quantitative measurement of GUCY2D , which could be adapted for diagnostic applications

• Develop standardized immunoassays for research and potential clinical use

These applications demonstrate how GUCY2D antibodies contribute to both basic science understanding and translational research in retinal diseases.

What methodological considerations apply when using GUCY2D antibodies in multiplex immunoassays?

Multiplex immunoassays, where multiple targets are detected simultaneously, require special considerations when incorporating GUCY2D antibodies:

Antibody Compatibility:

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

• For fluorescent multiplex assays, choose HRP-conjugated GUCY2D antibody with a distinct fluorophore from other targets

• Verify that epitope availability is not compromised by multiplexing conditions

Sequential Detection Strategies:

• For chromogenic IHC:

  • Use sequential detection rather than simultaneous incubation

  • Employ different substrates (DAB, Vector Red, etc.) for distinct visualization

  • Consider tyramide signal amplification for enhanced sensitivity and multiplexing capability

• For fluorescent applications:

  • Use HRP-conjugated GUCY2D antibody with tyramide-based amplification systems

  • Perform heat-mediated antibody stripping between rounds of detection

  • Carefully balance signal intensities across channels

Cross-Reactivity Prevention:

• Increase blocking stringency (5-10% normal serum from multiple species)

• Include detergents (0.1-0.3% Triton X-100) to reduce non-specific binding

• Test for cross-reactivity in single-stain controls before attempting multiplex

Optimized Detection Sequence:

• Detect lower abundance targets (potentially GUCY2D in disease states) first

• Follow with more abundant targets

• For each round of detection, include appropriate positive and negative controls

Validation of Multiplex Results:

• Confirm multiplex findings with single-target detection

• Use alternative methods (e.g., Western blot) to validate key findings

• Include appropriate controls for antibody specificity in multiplex context

These methodological considerations ensure reliable and interpretable results when incorporating GUCY2D antibodies into complex multiplex immunoassays for retinal research applications.