GUCY1A1 Antibody, FITC conjugated

What is GUCY1A1 and why is it important in biological research?

GUCY1A1 (Guanylate Cyclase 1 Soluble Subunit Alpha) is a critical component of the soluble guanylyl cyclase (sGC) enzyme complex. This heterodimeric enzyme consists of an alpha and beta subunit and functions as the primary receptor for nitric oxide (NO) in the NO-cGMP signaling pathway. When NO binds to the heme group associated with sGC, it activates the enzyme, catalyzing the conversion of GTP to cGMP, which acts as a second messenger in various physiological processes including vasodilation, neurotransmission, and platelet aggregation inhibition . The importance of GUCY1A1 in research stems from its central role in cardiovascular pathophysiology, neurological function, and various disease states where NO signaling is implicated, making antibodies against GUCY1A1 valuable tools for investigating these pathways.

What are the specific characteristics of FITC-conjugated GUCY1A1 antibodies?

FITC-conjugated GUCY1A1 antibodies typically combine the specificity of anti-GUCY1A1 immunoglobulins with the fluorescent properties of fluorescein isothiocyanate. These antibodies are commonly produced in rabbit hosts and are predominantly polyclonal in nature, though monoclonal versions exist for some epitopes . The FITC conjugation allows direct visualization of GUCY1A1 in fluorescence-based applications without requiring secondary antibodies. These antibodies are typically supplied at concentrations around 0.7 μg/μl in stabilization buffer and require storage at -20°C for long-term preservation of both antibody integrity and fluorophore activity . The excitation maximum for FITC is approximately 495 nm, with emission at 519 nm, placing it in the green spectrum of visible light.

What is the difference between GUCY1A1, GUCY1A3, and other guanylyl cyclase nomenclature?

The nomenclature surrounding guanylyl cyclases can be confusing due to historical naming conventions and updates in gene annotation. GUCY1A1 (previously known as GUCY1A3 in some databases) refers specifically to the alpha-1 subunit of soluble guanylyl cyclase. Other related proteins include GUCY1A2 (alpha-2 subunit), GUCY1B1 (beta-1 subunit), and GUCY1B3 (beta-3 subunit) . These different subunits combine to form functional heterodimeric enzymes with varying tissue distributions and regulatory properties. In contrast, membrane-bound guanylyl cyclases (particulate GCs) are single transmembrane proteins (e.g., GC-A, GC-B) that respond to peptide ligands rather than NO. When selecting antibodies for research, it's crucial to verify the specific target nomenclature to ensure experiments address the intended guanylyl cyclase component, as cross-reactivity between subunits can complicate interpretation of results.

What are the validated applications for FITC-conjugated GUCY1A1 antibodies?

FITC-conjugated GUCY1A1 antibodies have been validated for multiple experimental applications, with varying optimal dilutions for each technique. For Western blotting (WB), these antibodies can detect denatured GUCY1A1 protein at dilutions of approximately 1:10,000, making them highly sensitive for protein expression analysis . For immunoprecipitation (IP), a dilution of 1:200 is typically recommended to effectively capture the native protein complex . In enzyme-linked immunosorbent assays (ELISA), these antibodies perform well at dilutions around 1:500 . Additionally, they are suitable for immunohistochemistry (IHC) and immunofluorescence microscopy, where their direct FITC conjugation eliminates the need for secondary antibody incubation steps, simplifying protocols and reducing background signal. Some formulations have also been validated for flow cytometry and immunocytochemistry (ICC), particularly those developed with specific epitope targets within amino acids 22-214 of the protein .

How should I optimize immunofluorescence protocols using FITC-conjugated GUCY1A1 antibodies?

Optimizing immunofluorescence protocols with FITC-conjugated GUCY1A1 antibodies requires careful attention to several key parameters. First, fixation method significantly impacts epitope accessibility—4% paraformaldehyde (PFA) for 10-15 minutes at room temperature generally preserves GUCY1A1 antigenicity while maintaining cellular architecture. For membrane permeabilization, 0.1-0.3% Triton X-100 for 5-10 minutes typically provides adequate access to intracellular GUCY1A1 without excessive membrane disruption.

The blocking step is crucial to minimize non-specific binding; use 5-10% normal serum (derived from the same species as the secondary antibody, if used) with 1% BSA in PBS for 30-60 minutes at room temperature. For primary antibody incubation, begin with a 1:500 dilution of the FITC-conjugated GUCY1A1 antibody , then perform a dilution series (1:250, 1:500, 1:1000) to determine optimal signal-to-noise ratio for your specific sample type.

Since FITC is sensitive to photobleaching, minimize exposure to light during all steps, use antifade mounting media containing DAPI for nuclear counterstaining, and optimize microscope settings (exposure time, gain) to capture images before significant photobleaching occurs. Include appropriate controls: a no-primary antibody control to assess autofluorescence and non-specific secondary binding, and if possible, a GUCY1A1-knockout or siRNA-treated sample as a negative control.

What are the recommended sample preparation methods for Western blotting with GUCY1A1 antibodies?

For optimal Western blotting results with GUCY1A1 antibodies, proper sample preparation is essential. Begin by lysing cells or tissues in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors and phosphatase inhibitors if phosphorylation status is relevant. For tissues expressing GUCY1A1, such as vascular tissue, brain, or lung, homogenization should be performed quickly on ice to prevent protein degradation.

Protein quantification using BCA or Bradford assay should follow, with 20-50 μg of total protein typically loaded per lane. GUCY1A1 has a molecular weight of approximately 77-82 kDa, requiring an 8-10% polyacrylamide gel for adequate resolution . Heat samples at 95°C for 5 minutes in Laemmli buffer containing 5% β-mercaptoethanol for complete denaturation.

After electrophoresis, proteins should be transferred to PVDF membrane (preferred over nitrocellulose for GUCY1A1) using wet transfer (25V overnight at 4°C) for complete transfer of this higher molecular weight protein. For immunodetection, block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature, then incubate with GUCY1A1 antibody at 1:10,000 dilution . If using a non-conjugated primary antibody, follow with an appropriate HRP-conjugated secondary antibody and detect using enhanced chemiluminescence. For FITC-conjugated antibodies, fluorescence detection systems are required instead of chemiluminescence.

How can I verify the specificity of my GUCY1A1 antibody and avoid cross-reactivity issues?

Verifying antibody specificity is crucial for generating reliable data with GUCY1A1 antibodies. Several validation approaches should be employed in combination. First, perform Western blot analysis using positive control lysates from tissues known to express high levels of GUCY1A1 (e.g., lung, brain, vascular tissue) alongside negative controls where expression is minimal or absent. The antibody should detect a band at the expected molecular weight of approximately 77-82 kDa .

To evaluate potential cross-reactivity with other guanylyl cyclase subunits (particularly GUCY1A2, which shares sequence homology), perform peptide competition assays where the antibody is pre-incubated with excess immunizing peptide before application to samples. Signal abolishment indicates specificity for the target epitope. RNA interference approaches provide another validation method—compare staining patterns between samples with normal GUCY1A1 expression and those where GUCY1A1 has been knocked down using siRNA or shRNA.

For immunohistochemistry applications, compare staining patterns with published literature and use knockout tissue sections when available as definitive negative controls. Multiple antibodies targeting different epitopes of GUCY1A1 should show similar staining patterns if they are specific. Some GUCY1A1 antibodies have been specifically developed against synthetic peptides from particular regions (e.g., amino acids 654-704) , which can increase specificity but may limit applications depending on protein folding, processing, or post-translational modifications in your experimental system.

What are common sources of background or false positives when using FITC-conjugated antibodies, and how can they be minimized?

When working with FITC-conjugated GUCY1A1 antibodies, several factors can contribute to background or false positive signals. Autofluorescence is a primary concern, particularly in tissues rich in lipofuscin (such as brain, heart), collagen, or elastin, which naturally emit green fluorescence overlapping with FITC emission spectra. To mitigate this, pretreat sections with Sudan Black B (0.1-0.3% in 70% ethanol) for 10 minutes prior to immunostaining, or use spectral unmixing during image acquisition if available.

Inadequate blocking is another common issue—increase blocking time (1-2 hours) and concentration (5-10% serum with 1% BSA) to reduce non-specific binding. If tissue-specific background persists, adding 2% of the host species serum of your tissue to the antibody diluent can help.

For FITC-conjugated antibodies specifically, photobleaching can create inconsistent results. Minimize exposure to light during all protocol steps, prepare samples immediately before imaging when possible, and use antifade mounting media containing compounds like DABCO or propyl gallate.

Over-fixation can increase autofluorescence and mask epitopes—optimize fixation time and consider antigen retrieval methods if signal is weak. If background persists despite these measures, further antibody dilution (e.g., 1:1000 instead of 1:500) combined with longer incubation times (overnight at 4°C) often improves signal-to-noise ratio .

As a final quality control measure, always include a "no primary antibody" control processed identically to experimental samples to distinguish true signal from artifacts of sample processing or secondary antibody binding.

How stable is the FITC conjugation, and what are the optimal storage conditions to maintain antibody performance?

The stability of FITC conjugation to GUCY1A1 antibodies is influenced by several factors that researchers should carefully manage. FITC conjugates are generally stable for 12-18 months when properly stored, but their fluorescence activity gradually decreases over time due to photobleaching and hydrolysis of the thiourea bond between FITC and antibody proteins. To maintain optimal performance, FITC-conjugated GUCY1A1 antibodies should be stored at -20°C for long-term storage in the dark . Avoid repeated freeze-thaw cycles, which can accelerate degradation—aliquot antibodies upon receipt into single-use volumes.

The storage buffer composition significantly impacts stability. Most commercial FITC-conjugated GUCY1A1 antibodies are supplied in stabilization buffers containing protein (BSA or gelatin) at approximately 0.7 μg/μl, with preservatives like sodium azide (0.05%) to prevent microbial growth . The optimal pH range for FITC conjugates is 7.2-8.0; more acidic conditions can accelerate fluorophore degradation.

For working solutions during experiments, maintain antibodies on ice and protected from light. If experimental protocols require diluted antibody storage, short-term refrigeration (2-8°C for up to one week) is preferable to freezing diluted solutions, which can cause protein precipitation and fluorophore quenching. To monitor potential degradation, researchers should periodically compare the performance of stored antibodies against fresh lots using positive control samples with known GUCY1A1 expression patterns.

How can FITC-conjugated GUCY1A1 antibodies be utilized in multi-parameter flow cytometry?

FITC-conjugated GUCY1A1 antibodies can be effectively incorporated into multi-parameter flow cytometry panels for comprehensive analysis of the NO-cGMP signaling pathway in various cell populations. When designing such panels, strategic fluorophore selection is essential since FITC's emission spectrum (peak ~519 nm) overlaps with PE, creating compensation challenges. Ideally, pair FITC-GUCY1A1 with fluorophores having minimal spectral overlap such as APC, APC-Cy7, or BV421.

For intracellular detection of GUCY1A1, optimization of permeabilization protocols is crucial. While saponin-based buffers (0.1-0.3%) preserve most surface epitopes while allowing intracellular access, methanol fixation may provide superior results for cytosolic proteins like GUCY1A1, though it can distort some surface markers. A sequential staining approach often works best—first stain surface markers, then fix, permeabilize, and stain for GUCY1A1.

For co-expression studies examining GUCY1A1 with its dimerization partners (GUCY1B1 or GUCY1B3), or with downstream signaling molecules like protein kinase G (PKG), carefully titrate each antibody to minimize spillover. Typical starting dilutions for FITC-GUCY1A1 in flow cytometry range from 1:200 to 1:500 , but optimization for each cell type is essential.

Creating biologically relevant gates requires appropriate controls: fluorescence-minus-one (FMO) controls help set boundaries between positive and negative populations, while biological controls comparing high-expressing cells (vascular smooth muscle cells, platelets) with low-expressing cells help validate staining specificity. For phospho-flow applications examining GUCY1A1 activation state in response to NO donors or pharmacological agents, rapid fixation with 4% PFA followed by permeabilization with 90% methanol often provides optimal epitope preservation.

What approaches can be used to study GUCY1A1 protein-protein interactions using FITC-conjugated antibodies?

FITC-conjugated GUCY1A1 antibodies enable several sophisticated approaches for investigating protein-protein interactions within the soluble guanylyl cyclase signaling complex. Proximity ligation assay (PLA) represents one powerful technique where FITC-conjugated anti-GUCY1A1 can be paired with antibodies against potential interaction partners (such as GUCY1B1, HSP90, or PDE enzymes). After primary antibody binding, species-specific secondary antibodies conjugated with complementary oligonucleotides allow amplification and fluorescent detection when proteins are within 40nm of each other, providing spatial resolution of interactions in situ.

Fluorescence resonance energy transfer (FRET) microscopy can be employed when pairing FITC-GUCY1A1 (donor) with antibodies against interaction partners conjugated to appropriate acceptor fluorophores (such as TRITC or Cy3). Energy transfer between fluorophores occurs only when proteins are within 10nm, providing strong evidence of direct molecular interaction. The FRET efficiency can be quantified through acceptor photobleaching or spectral unmixing approaches.

For co-immunoprecipitation studies, FITC-GUCY1A1 antibodies can be immobilized on anti-FITC conjugated magnetic beads, allowing gentle isolation of intact protein complexes while minimizing background often seen with protein A/G approaches . The precipitated complexes can then be analyzed by Western blotting or mass spectrometry to identify novel interaction partners.

Additionally, fluorescence recovery after photobleaching (FRAP) using FITC-GUCY1A1 antibodies in live cells (delivered via cell-penetrating peptides) can provide insights into protein mobility and how it changes upon complex formation following NO donor stimulation or pharmacological intervention.

How can I quantitatively measure GUCY1A1 expression levels in different tissue samples?

Quantitative assessment of GUCY1A1 expression across tissue samples requires careful method selection and rigorous standardization. For protein-level quantification, quantitative Western blotting represents a reliable approach. Prepare tissue lysates with standardized protein extraction protocols, load equal amounts (validated by housekeeping proteins like GAPDH or β-actin), and use GUCY1A1 antibodies at optimized dilutions (1:10,000 is typically recommended) . Include a standard curve of recombinant GUCY1A1 protein at known concentrations to enable absolute quantification. Densitometric analysis should normalize GUCY1A1 signal to loading controls.

For higher throughput analysis, sandwich ELISA provides reliable quantification. Commercial kits are available, or researchers can develop custom assays using capture antibodies against one GUCY1A1 epitope and FITC-conjugated detection antibodies against a different epitope, with signal measured via fluorescence plate readers. Typical working dilutions for ELISA applications range from 1:500 to 1:1000 .

In tissue sections, quantitative immunofluorescence allows spatial assessment of expression patterns. Use identical acquisition parameters (exposure time, gain) for all samples, and include fluorescent calibration beads in each imaging session to normalize between experiments. For tissue microarrays or large sample sets, automated image analysis software enables unbiased quantification of FITC-GUCY1A1 signal intensity, with cell-type specific markers to differentiate expression in distinct populations.

For absolute quantification, mass spectrometry-based approaches like selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) provide ultimate specificity and sensitivity, though these require specialized equipment. GUCY1A1 antibodies can be used for immunoaffinity enrichment prior to MS analysis, enhancing detection of low-abundance GUCY1A1 in complex tissue samples.

How can FITC-conjugated GUCY1A1 antibodies be used in super-resolution microscopy?

FITC-conjugated GUCY1A1 antibodies can be effectively adapted for super-resolution microscopy techniques, though certain optimizations are necessary to overcome the inherent limitations of FITC as a fluorophore. For structured illumination microscopy (SIM), which achieves approximately 120 nm resolution, standard immunofluorescence protocols using FITC-GUCY1A1 antibodies can be applied with minor modifications. Use high-quality No. 1.5H coverslips (170 ± 5 μm thickness) and mounting media with matched refractive index to minimize spherical aberrations. Since SIM requires high signal-to-noise ratios, more concentrated antibody solutions (approximately 1:250 dilution) may be necessary compared to conventional microscopy .

Stimulated emission depletion (STED) microscopy can achieve 30-80 nm resolution with FITC, though with reduced efficiency compared to dyes specifically optimized for STED. When using FITC-GUCY1A1 in STED applications, increasing laser power may be necessary, potentially accelerating photobleaching. Consider photoprotectant mounting media containing oxygen-scavenging systems to extend imaging time.

For all super-resolution techniques, sample preparation quality becomes even more critical. Careful fixation (2% PFA without methanol), thorough blocking (overnight at 4°C with 5% BSA, 5% normal serum, 0.1% Triton X-100), and extended antibody incubation times (24-48 hours at 4°C) often improve labeling density and specificity at the nanoscale level.

What are the considerations for using GUCY1A1 antibodies in high-throughput screening applications?

Adapting FITC-conjugated GUCY1A1 antibodies for high-throughput screening (HTS) applications requires careful optimization to balance throughput, sensitivity, and specificity. For cell-based HTS in microplate formats, several key considerations apply. First, antibody concentration must be optimized through checkerboard titration across multiple cell densities to identify conditions that provide robust signal-to-background ratio while minimizing reagent consumption. Starting dilutions of 1:500 are recommended , but verification across the specific cell types in your screening panel is essential.

Fixation and permeabilization protocols must be adapted for automation compatibility—paraformaldehyde (2-4%) fixation for 10-15 minutes followed by brief (5 minute) permeabilization with 0.1% Triton X-100 typically provides good results for intracellular GUCY1A1 detection while minimizing processing steps. Consider one-step fixation/permeabilization reagents for further streamlining.

For assay miniaturization to 384 or 1536-well formats, antibody incubation volumes and times require optimization. Typically, reducing volumes while extending incubation times (e.g., 4°C overnight) maintains sensitivity while reducing reagent costs. Specialized microplate readers with appropriate excitation (488 nm) and emission (520 nm) filters are required for FITC detection, with consideration of potential well-to-well crosstalk in high-density plate formats.

Z'-factor assessment using positive controls (NO donor-stimulated cells with high GUCY1A1 expression) and negative controls (siRNA-knockdown or tissues known to express minimal GUCY1A1) should achieve values >0.5 for reliable screening. Multiplexing opportunities exist by pairing FITC-GUCY1A1 with antibodies against downstream pathway components (such as PKG or phosphorylated targets) labeled with spectrally distinct fluorophores, enabling simultaneous pathway analysis in a single well.

What are the comparative properties of different commercially available GUCY1A1 antibodies?

Table compiled from multiple commercial sources and technical specifications. Specific product characteristics may vary by manufacturer.

What is the dilution range optimization table for FITC-conjugated GUCY1A1 antibodies across applications?

Based on technical data from product datasheets and research literature. Optimal dilutions should be determined for each experimental system.

What biological samples are most suitable for GUCY1A1 antibody applications?

Table compiled from expression databases and literature reports. Expression levels may vary with physiological/pathological conditions.

What are emerging trends in GUCY1A1 antibody development and applications?

The development and application of GUCY1A1 antibodies is evolving rapidly with several emerging trends that will expand their utility in research. Recombinant antibody technology is increasingly being applied to generate highly specific monoclonal antibodies against defined GUCY1A1 epitopes with reduced batch-to-batch variability compared to traditional polyclonal approaches . These recombinant antibodies offer superior reproducibility for quantitative applications and long-term studies.

Multiplexing capabilities are advancing through development of GUCY1A1 antibodies conjugated to spectrally distinct fluorophores beyond FITC, including far-red emitting dyes that minimize tissue autofluorescence interference and facilitate deep-tissue imaging. Furthermore, novel conjugation chemistries that improve fluorophore stability and brightness are enhancing sensitivity for detecting low-abundance GUCY1A1 in challenging samples.

Single-domain antibodies (nanobodies) against GUCY1A1 are beginning to emerge, offering smaller size (approximately 15 kDa versus 150 kDa for conventional antibodies) for superior tissue penetration and epitope access in densely packed cellular structures. These nanobodies show particular promise for super-resolution microscopy applications where the distance between fluorophore and epitope critically impacts achievable resolution.

Integration with CRISPR-based technologies represents another frontier, where GUCY1A1 antibodies are being combined with proximity-dependent enzymes to enable targeted genomic or epigenomic modifications at GUCY1A1-associated loci. Additionally, computational approaches for in silico antibody engineering are accelerating development of GUCY1A1 antibodies with optimized binding properties for specific research applications.