GUCY1A1 Antibody, Biotin conjugated

What is GUCY1A1 and what cellular functions does it regulate?

GUCY1A1 (Guanylate Cyclase 1 Soluble Subunit Alpha 1) is a protein-coding gene that encodes an alpha subunit of the soluble guanylate cyclase (sGC) complex. The protein interacts with a beta subunit to form the functional guanylate cyclase enzyme, which catalyzes the conversion of GTP to 3',5'-cyclic GMP and pyrophosphate upon activation by nitric oxide. This enzyme plays a critical role in cellular signaling pathways, particularly those involving vasodilation, platelet aggregation inhibition, and neurotransmission. Several transcript variants encoding different isoforms have been identified for this gene. GUCY1A1 is also known by other names including GUCA3, MYMY6, GC-SA3, GUC1A3, GUCSA3, GUCY1A3, GCS-alpha-3, and GC-S-alpha-1 .

What applications are GUCY1A1 antibodies typically used for in research?

GUCY1A1 antibodies have diverse applications in research protocols. The most common applications include:

• Western Blotting (WB): For detection of denatured GUCY1A1 protein in tissue or cell lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of GUCY1A1 in solution

• Immunohistochemistry (IHC): For localization of GUCY1A1 in tissue sections

• Immunofluorescence (IF): For cellular localization studies

• Flow Cytometry (FACS): For detecting GUCY1A1 in individual cells

• Immunoprecipitation (IP): For isolation of GUCY1A1 protein complexes

The biotin-conjugated variant specifically offers enhanced detection sensitivity when used with streptavidin-based detection systems, making it particularly valuable for applications requiring signal amplification .

What are the key specifications of commercially available biotin-conjugated GUCY1A1 antibodies?

Most commercially available biotin-conjugated GUCY1A1 antibodies share the following specifications:

These antibodies are typically raised against specific amino acid sequences within GUCY1A1, most commonly the 20-152 AA region, which represents a conserved functional domain of the protein .

What are the optimal sample preparation methods for GUCY1A1 detection in different tissues?

Sample preparation for GUCY1A1 detection varies by tissue type and application:

For kidney, heart, lung, and liver tissues (where GUCY1A1 specifically marks fibroblasts):

• Fixation:

  • Fresh tissues: 4% paraformaldehyde for 24 hours at 4°C

  • Flash freezing in liquid nitrogen for cryosections

• Sectioning:

  • Paraffin-embedded: 5-7 μm sections optimal for IHC/IF

  • Cryosections: 8-10 μm sections for maintained antigenicity

• Antigen Retrieval (critical for GUCY1A1):

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

  • Enzymatic retrieval with proteinase K may preserve tissue architecture better

• Permeabilization:

  • 0.1-0.3% Triton X-100 in PBS for 10-15 minutes

  • Critical for accessing intracellular GUCY1A1 epitopes

For cellular preparations:

• Fixation with 4% paraformaldehyde for 15 minutes at room temperature

• Gentle permeabilization with 0.1% Triton X-100 for 5-10 minutes

These methods have been validated in studies examining GUCY1A1 as a fibroblast marker in multiple organs and are essential for accessing the target epitopes while preserving tissue morphology .

How can researchers optimize blocking conditions for GUCY1A1 immunodetection?

Optimizing blocking conditions is critical for reducing background and enhancing specific GUCY1A1 detection:

• For biotin-conjugated antibody applications:

  • An avidin/biotin blocking step is essential to block endogenous biotin

  • Commercial avidin/biotin blocking kits are recommended (15 minutes incubation with avidin followed by 15 minutes with biotin)

• Primary blocking considerations:

  • 5-10% normal serum (from species unrelated to primary antibody host)

  • 1-5% BSA in PBS or TBS (higher concentrations for tissues with high background)

  • Addition of 0.1-0.3% Triton X-100 for membrane permeabilization

  • Blocking for 1-2 hours at room temperature or overnight at 4°C

• Tissue-specific adjustments:

  • Kidney tissue: Add 0.05% Tween-20 to reduce tubular background

  • Liver tissue: Extended blocking (2+ hours) and higher serum concentration (10%)

  • Heart and lung: Addition of 0.1% fish gelatin can reduce non-specific binding

• Additional components to consider:

  • 0.05-0.1% Tween-20 to reduce hydrophobic interactions

  • 0.1-0.3% cold fish skin gelatin as an alternative protein blocker

  • 5-10 mM glycine to block free aldehyde groups from fixation

These optimizations have been derived from experimental protocols where GUCY1A1 has been successfully detected in multiple tissue types and help ensure specific binding while minimizing background interference .

How can GUCY1A1 antibodies be used to investigate fibrosis in multiple organ systems?

GUCY1A1 has been identified as a specific marker for fibroblasts in kidney, heart, lung, and liver tissues, making it a valuable tool for fibrosis research across multiple organ systems. Research strategies include:

• Kidney fibrosis investigation:

  • GUCY1A1 comprehensively labels both quiescent and activated fibroblasts in the kidney cortex and medulla

  • Unlike traditional markers (Pdgfrβ and vimentin), GUCY1A1 does not overlap with off-target populations like podocytes

  • GUCY1A1 levels significantly increase during chronic kidney disease (CKD) progression and correlate with established fibrosis markers

  • Detection works effectively in both male and female experimental models

• Cardiac fibrosis assessment:

  • GUCY1A1 can be used to track activated fibroblasts during cardiac injury, progression, and resolution

  • Co-staining with Pdgfrβ, vimentin, and αSma allows identification of different fibroblast activation states

• Liver fibrosis monitoring:

  • GUCY1A1 expression increases in activated hepatic stellate cells and fibroblasts

  • Parallels injury progression and resolution in experimental models

• Comparative multi-organ analysis:

  • Enables standardized fibroblast identification across different organs for comparative fibrosis studies

  • Facilitates investigation of common and tissue-specific fibrosis mechanisms

This approach allows for consistent cross-organ comparison of fibroblast activity and fibrotic progression using a single marker, which has significant advantages over traditional multi-marker panels that can vary in specificity between different tissues .

What are the most effective multiplex staining strategies when using biotin-conjugated GUCY1A1 antibodies?

Effective multiplex staining with biotin-conjugated GUCY1A1 antibodies requires careful planning to avoid cross-reactivity and signal interference:

• Sequential detection approach:

  • Complete GUCY1A1 detection with streptavidin-conjugated fluorophore first

  • Block remaining biotin binding sites with excess unconjugated streptavidin

  • Proceed with subsequent antibody staining using directly conjugated primary antibodies or alternative detection systems

• Recommended marker combinations for fibrosis research:

  • GUCY1A1 (biotin/streptavidin detection) + αSMA (direct fluorophore conjugate) + PDGFRβ (alternative host species with secondary detection)

  • This combination allows distinction between quiescent fibroblasts (GUCY1A1+/αSMA-/PDGFRβ+) and myofibroblasts (GUCY1A1+/αSMA+/PDGFRβ+)

• Tyramide signal amplification (TSA) integration:

  • For weak GUCY1A1 signals, incorporate TSA with HRP-streptavidin

  • Complete this reaction first, heat-inactivate HRP, then proceed with conventional staining

  • Allows for amplification of GUCY1A1 signal while maintaining multiplex capability

• Spectral unmixing considerations:

  • Choose fluorophores with minimal spectral overlap for multiplexing

  • Consider far-red fluorophores for streptavidin conjugation to minimize tissue autofluorescence interference

  • Use single-stained controls for accurate spectral unmixing

These strategies enable researchers to effectively combine GUCY1A1 detection with other fibroblast or cell-type markers for comprehensive analysis of tissue remodeling and fibrosis progression across multiple organ systems .

What analytical approaches are recommended for quantifying GUCY1A1 expression in tissue samples?

Quantification of GUCY1A1 expression in tissues requires robust analytical approaches:

• Digital image analysis for IHC/IF quantification:

  • Threshold-based segmentation of GUCY1A1+ cells using ImageJ/FIJI or similar software

  • Automated counting of positive cells per high-power field or tissue area

  • Colocalization analysis with other markers using Manders' or Pearson's correlation coefficients

  • Intensity measurement to assess relative expression levels between experimental groups

• Western blot quantification:

  • Normalization to housekeeping proteins (β-actin, GAPDH) with correction for loading variations

  • Densitometric analysis using standard curve of recombinant protein for absolute quantification

  • Semi-quantitative comparison between sample groups with statistical validation

• ELISA-based quantification:

  • Standard curve preparation using recombinant GUCY1A1 protein

  • Sample dilution optimization to ensure readings within linear range

  • Calculation of GUCY1A1 concentration using four-parameter logistic regression

• Tissue-specific considerations:

  • Kidney: Separate analysis of cortical and medullary regions

  • Heart: Adjustment for cardiomyocyte density differences between regions

  • Lung: Correction for airspace in quantification of interstitial fibroblasts

  • Liver: Zonation-specific analysis (periportal vs. pericentral)

• Correlation with disease progression:

  • Integration of GUCY1A1 quantification with clinical parameters

  • Correlation analysis with established fibrosis markers (α-SMA, collagen deposition)

  • Time-course analysis to establish temporal relationship with disease progression

These analytical approaches enable accurate quantification of GUCY1A1 expression across experimental conditions and tissue types, facilitating comparison between studies and meaningful interpretation of results in the context of fibrosis progression .

What are the most common causes of non-specific binding when using GUCY1A1 antibodies and how can they be resolved?

Non-specific binding is a common challenge with GUCY1A1 antibodies. The main causes and solutions include:

• Endogenous biotin interference:

  • Problem: Tissues like kidney, liver, and brain contain high levels of endogenous biotin

  • Solution: Implement avidin/biotin blocking step prior to antibody incubation

  • Alternative: Consider using non-biotin detection systems for tissues with high endogenous biotin

• Insufficient blocking:

  • Problem: Inadequate blocking leads to non-specific antibody binding

  • Solution: Extend blocking time (2+ hours or overnight) and increase blocker concentration

  • Alternative: Try different blocking agents (BSA, normal serum, casein) to determine optimal formulation

• Cross-reactivity with related proteins:

  • Problem: GUCY1A1 shares sequence homology with other guanylate cyclase subunits

  • Solution: Verify antibody specificity using knockout controls or competing peptides

  • Alternative: Use antibodies targeting unique epitopes (e.g., AA 20-152 region) with validated specificity

• Suboptimal antibody concentration:

  • Problem: Too high concentration increases background staining

  • Solution: Perform titration experiments to determine optimal antibody dilution for each application

  • Recommendation: Start with higher dilutions (1:500-1:1000) and adjust based on signal-to-noise ratio

• Tissue fixation artifacts:

  • Problem: Overfixation can cause epitope masking and increase non-specific binding

  • Solution: Optimize fixation time and implement effective antigen retrieval protocols

  • Alternative: Test both frozen and paraffin-embedded samples to determine optimal preparation

By systematically addressing these issues, researchers can significantly improve the specificity of GUCY1A1 antibody staining across different experimental conditions and tissue types .

How can researchers validate GUCY1A1 antibody specificity and performance?

Validating GUCY1A1 antibody specificity is crucial for reliable research outcomes. A comprehensive validation approach includes:

• Genetic controls:

  • Positive control: Overexpression systems (e.g., transfected cells)

  • Negative control: GUCY1A1 knockout tissues or siRNA-mediated knockdown cells

  • Zebrafish models with gucy1a1 knockout provide excellent validation systems

• Peptide competition assays:

  • Pre-incubation of antibody with excess immunizing peptide should abolish specific staining

  • Gradual peptide concentration titration can determine binding affinity

• Multi-technique validation:

  • Orthogonal validation using different detection methods (IHC, WB, IF)

  • Consistent results across techniques strongly support antibody specificity

• Cross-species reactivity assessment:

  • Test antibody in multiple species with known sequence homology

  • Expected patterns based on evolutionary conservation provide validation

• Western blot molecular weight verification:

  • Confirm band at expected molecular weight (~77-82 kDa for GUCY1A1)

  • Multiple bands may represent splice variants (documented in database)

• Lot-to-lot consistency testing:

  • Maintain reference samples to test each new antibody lot

  • Document staining patterns, intensity, and background for comparison

• Positive control tissues:

  • Kidney fibroblasts show strong, consistent GUCY1A1 expression

  • Heart, lung, and liver fibroblasts also provide reliable positive controls

A comprehensive validation using multiple approaches ensures confidence in antibody performance and experimental results, particularly important for GUCY1A1 given its role as a specific fibroblast marker across multiple tissues .

What are the best approaches for resolving weak or absent signals in GUCY1A1 detection?

When faced with weak or absent GUCY1A1 signals, researchers can implement several optimization strategies:

• Antigen retrieval optimization:

  • Test multiple methods: heat-induced (citrate, EDTA, Tris buffers), enzymatic (proteinase K, trypsin)

  • Optimize retrieval time and temperature (e.g., 95-100°C for 10-30 minutes)

  • For tissues with dense ECM (fibrotic samples), extend retrieval time by 5-10 minutes

• Signal amplification techniques:

  • Implement avidin-biotin complex (ABC) method for enhanced sensitivity

  • Use tyramide signal amplification (TSA) for fluorescent applications

  • Consider polymer-based detection systems for chromogenic applications

• Tissue preparation adjustments:

  • Reduce fixation time to prevent excessive crosslinking

  • Test cryopreservation as alternative to paraffin embedding

  • Process samples rapidly to minimize protein degradation

• Antibody incubation optimization:

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

  • Reduce washing stringency (shorter washes, lower detergent concentration)

  • Test different antibody diluents (with protein carriers or enhancers)

• Detection system considerations:

  • For biotin-conjugated antibodies, ensure fresh streptavidin-HRP/fluorophore

  • Extend substrate development time for chromogenic detection

  • Use high-sensitivity fluorophores (quantum dots, Alexa Fluor 647) for IF

• Sample-specific adjustments:

  • For highly fibrotic tissues, increase permeabilization

  • For samples with low GUCY1A1 expression, concentrate protein lysates for Western blotting

  • For ELISA, reduce sample dilution to increase target concentration

These strategies can significantly improve detection sensitivity and should be systematically tested when troubleshooting weak GUCY1A1 signals in experimental samples .

How does GUCY1A1 expression change in cardiovascular disease models?

GUCY1A1 expression undergoes significant changes in cardiovascular disease models, providing insights into pathophysiological mechanisms:

• Hypertension models:

  • GUCY1A1 genetic variants have been associated with blood pressure regulation through genome-wide association studies

  • Zebrafish gucy1a1 knockout models demonstrate altered blood flow dynamics and vascular response

  • Expression changes correlate with alterations in NO-cGMP signaling pathway activity

• Coronary artery disease models:

  • GWAS studies have identified GUCY1A1 as a susceptibility locus for coronary artery disease

  • Expression changes occur in vascular smooth muscle cells during atherosclerotic progression

  • Altered GUCY1A1 function affects vascular tone and remodeling

• Cardiac fibrosis and remodeling:

  • GUCY1A1 marks activated cardiac fibroblasts during injury and remodeling

  • Expression increases parallel myofibroblast activation and correlates with extracellular matrix deposition

  • Temporal expression patterns follow injury progression and resolution phases

• Physiological vs. pathological expression:

  • Basal expression maintains normal vascular responsiveness to nitric oxide

  • Altered expression in disease states affects downstream signaling pathways

  • Compensatory upregulation may occur in early disease stages followed by potential downregulation in advanced disease

These expression patterns provide valuable biomarkers for disease progression and potential therapeutic targets for cardiovascular conditions, highlighting the importance of GUCY1A1 antibodies in cardiovascular research .

What is the significance of GUCY1A1 in kidney fibrosis research?

GUCY1A1 has emerged as a particularly valuable marker in kidney fibrosis research due to several unique characteristics:

• Superior specificity compared to traditional markers:

  • GUCY1A1 specifically marks kidney fibroblasts without labeling other cell populations

  • Unlike PDGFRβ and vimentin, GUCY1A1 does not cross-react with podocytes or other renal cell types

  • This specificity enables more accurate quantification of fibroblast populations

• Dynamic expression during disease progression:

  • GUCY1A1 levels significantly increase during chronic kidney disease (CKD) progression

  • Expression levels directly correlate with established fibrosis markers

  • Temporal expression changes parallel fibrotic tissue remodeling

• Comprehensive fibroblast labeling:

  • Labels both cortical and medullary fibroblasts

  • Marks both quiescent fibroblasts in healthy kidneys and activated fibroblasts during injury

  • Works effectively in both male and female experimental models

• Translational relevance:

  • Elevated GUCY1A1 expression observed in human fibrotic kidney tissues

  • Potential therapeutic target for anti-fibrotic interventions

  • Biomarker potential for monitoring fibrosis progression and treatment response

• Experimental applications:

  • Enables accurate tracking of fibroblast activation during CKD progression

  • Facilitates lineage tracing studies to determine cellular origins of kidney fibroblasts

  • Allows precise quantification of fibroblast proliferation and ECM production

These characteristics make GUCY1A1 a valuable tool for investigating kidney fibrosis mechanisms, evaluating potential therapeutic interventions, and understanding the cellular dynamics of chronic kidney disease progression .

How can GUCY1A1 antibodies contribute to research on pulmonary and hepatic fibrosis?

GUCY1A1 antibodies offer significant advantages for researching fibrosis in both pulmonary and hepatic systems:

• Pulmonary fibrosis research applications:

  • Specific identification of lung fibroblasts without marking epithelial or immune cells

  • Monitoring fibroblast activation during idiopathic pulmonary fibrosis progression

  • Quantification of myofibroblast transformation in response to profibrotic stimuli

  • Evaluation of anti-fibrotic therapeutic efficacy in experimental models

  • Correlation with clinical parameters and other fibrosis markers

• Hepatic fibrosis research advantages:

  • Specific marking of hepatic stellate cells and portal fibroblasts during activation

  • Distinguishing between different fibroblast populations in liver injury models

  • Tracking fibroblast dynamics during fibrosis progression and resolution

  • Evaluating zonal differences in fibroblast activation across liver architecture

  • Assessment of therapeutic interventions targeting fibroblast activation

• Multi-organ comparative studies:

  • Using a single marker (GUCY1A1) across different organs enables direct comparison of fibrotic processes

  • Investigation of shared and organ-specific fibrosis mechanisms

  • Standardized quantification methods across tissue types

  • Identification of common therapeutic targets with multi-organ applications

• Clinical translation potential:

  • Assessment of GUCY1A1 as a biomarker for fibrosis progression

  • Evaluation as a therapeutic target for anti-fibrotic interventions

  • Correlation with treatment response and clinical outcomes

The ability to specifically mark fibroblasts across multiple organ systems makes GUCY1A1 antibodies particularly valuable for comprehensive fibrosis research, enabling more accurate characterization of fibrotic processes and potential therapeutic interventions in both pulmonary and hepatic diseases .

What emerging applications of GUCY1A1 antibodies show promise for translational research?

Several emerging applications of GUCY1A1 antibodies show significant promise for translational research:

• Fibrosis biomarker development:

  • Detection of circulating GUCY1A1+ fibroblasts or extracellular vesicles as liquid biopsy markers

  • Correlation of tissue GUCY1A1 expression with disease progression and treatment response

  • Development of non-invasive imaging probes targeting GUCY1A1 for fibrosis visualization

• Precision medicine applications:

  • Stratification of patients based on GUCY1A1 expression patterns

  • Identification of responders to anti-fibrotic therapies

  • Personalized treatment approaches based on fibroblast activation status

• Therapeutic targeting strategies:

  • GUCY1A1-targeted drug delivery systems for fibroblast-specific interventions

  • Antibody-drug conjugates for selective depletion of activated fibroblasts

  • Development of small molecule modulators of GUCY1A1 activity

• Regenerative medicine applications:

  • Monitoring fibroblast dynamics during tissue regeneration

  • Optimization of biomaterial scaffolds to modulate fibroblast activity

  • Guiding stem cell differentiation toward non-fibrotic healing

• Multi-omics integration:

  • Correlation of GUCY1A1 protein expression with transcriptomic and metabolomic profiles

  • Single-cell analysis of GUCY1A1+ cells to identify fibroblast subpopulations

  • Systems biology approaches to understand GUCY1A1 regulatory networks

These emerging applications leverage the specificity of GUCY1A1 as a fibroblast marker across multiple organs and have potential to significantly advance translational research in fibrosis-related diseases .

How might advanced genetic models enhance our understanding of GUCY1A1 function?

Advanced genetic models offer powerful approaches for elucidating GUCY1A1 function:

• Conditional knockout models:

  • Cell-specific GUCY1A1 deletion using Cre-lox systems

  • Temporal control of GUCY1A1 expression using inducible promoters

  • Investigation of organ-specific functions through tissue-restricted deletion

  • Assessment of developmental versus homeostatic roles

• CRISPR/Cas9 applications:

  • Generation of precise point mutations mimicking human disease variants

  • Knock-in of reporter genes for live tracking of GUCY1A1 expression

  • Creation of humanized GUCY1A1 models for translational studies

  • High-throughput screening of GUCY1A1 regulatory elements

• Zebrafish gucy1a1 models:

  • Transparent embryos allow visualization of cardiovascular development

  • Rapid generation of knockout lines for functional studies

  • Drug screening platforms for compounds targeting the NO-cGMP pathway

  • Study of evolutionary conservation of GUCY1A1 function

• Single-cell resolution approaches:

  • GUCY1A1 lineage tracing to determine cell fate during disease progression

  • Spatial transcriptomics to map GUCY1A1+ cell neighborhoods

  • Optogenetic control of GUCY1A1 activity in specific cell populations

  • In vivo imaging of GUCY1A1+ cells during disease processes

• Multi-organ systems:

  • Models with simultaneous monitoring of GUCY1A1 function across organs

  • Investigation of organ crosstalk mediated by GUCY1A1 signaling

  • Comparative analysis of tissue-specific regulatory mechanisms

These advanced genetic approaches promise to reveal new insights into GUCY1A1 biology, disease mechanisms, and potential therapeutic interventions, extending beyond traditional antibody-based detection to functional characterization .

What methodological advances may improve GUCY1A1 detection in challenging samples?

Several methodological advances show promise for enhancing GUCY1A1 detection in challenging samples:

• Advanced microscopy techniques:

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization

  • Expansion microscopy for physical magnification of tissue structures

  • Light-sheet microscopy for 3D visualization of intact organs

  • Multi-photon microscopy for deep tissue imaging with reduced phototoxicity

• Novel signal amplification approaches:

  • DNA-based signal amplification (RollAmp, HCR) for exponential signal enhancement

  • Proximity ligation assay for detecting protein-protein interactions with GUCY1A1

  • Click chemistry-based detection systems for reduced background

  • Lanthanide-based time-resolved fluorescence for improved signal-to-noise ratio

• Mass spectrometry imaging:

  • Imaging mass cytometry for multiplexed protein detection

  • MALDI-MSI for spatial proteomic analysis in tissue sections

  • Mass spectrometry-based immunohistochemistry for quantitative protein measurement

• AI-assisted image analysis:

  • Deep learning algorithms for automated fibroblast identification

  • Computer vision for quantitative assessment of GUCY1A1 expression patterns

  • Machine learning for correlation of expression with morphological features

• Sample preparation innovations:

  • Tissue clearing techniques for whole-organ GUCY1A1 visualization

  • Hydrogel-tissue chemistry for improved antigen preservation

  • Microfluidic tissue processing for standardized preparation

  • Cryopreservation methods optimized for membrane protein preservation

These methodological advances have the potential to significantly enhance the sensitivity, specificity, and quantitative nature of GUCY1A1 detection in challenging samples, such as highly fibrotic tissues, archived specimens, or samples with low expression levels .