GUCD1 Antibody, FITC conjugated

What is GUCD1 and what cellular functions is it associated with?

GUCD1 (Guanylyl cyclase domain-containing protein 1) is also known as Protein LLN4, identified by UniProt ID Q96NT3. It contains a guanylyl cyclase domain, suggesting potential involvement in cyclic GMP (cGMP) signaling pathways. The protein is encoded by a gene located on chromosome 22 (C22orf13). While less well-characterized than other guanylyl cyclase proteins, its domain structure suggests possible roles in signal transduction pathways mediated by cGMP-dependent mechanisms .

What is the difference between GUCY2C and GUCD1 antibodies?

Despite similar nomenclature, these antibodies target distinct proteins:

When selecting between these antibodies, researchers should carefully consider which protein is relevant to their specific research question .

What are the optimal storage conditions for FITC-conjugated GUCD1 antibody?

For optimal preservation of both antibody function and fluorophore integrity:

• Store at -20°C or -80°C for long-term preservation

• Avoid repeated freeze-thaw cycles which degrade both antibody and fluorophore

• Protect from light to prevent photobleaching of the FITC molecule

• When in lyophilized form, the antibody typically has greater stability

• Once reconstituted, aliquot to minimize freeze-thaw cycles

• The standard buffer contains 50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative

• Always follow the specific Certificate of Analysis (CoA) instructions for your particular lot

What applications is FITC-conjugated GUCD1 antibody validated for?

Based on available information, FITC-conjugated GUCD1 antibody has been primarily validated for:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Potentially useful for immunofluorescence applications, though validation for specific cell types or tissues should be performed

• Flow cytometry applications may be possible given the FITC conjugation

Researchers should note that applications beyond those specifically validated require end-user verification with appropriate controls.

What controls should be used when working with FITC-conjugated GUCD1 antibody?

Three essential types of controls are required for rigorous experiments:

Primary Antibody Controls (specificity verification):

• Ideally, use tissue/cells from a knockout organism lacking GUCD1

• Alternatively, use siRNA knockdown cells with reduced GUCD1 expression

• Perform immunoblots to confirm the antibody recognizes a single protein of correct molecular weight

• Consider absorption controls using purified GUCD1 protein to confirm binding specificity

Secondary Antibody Controls (if using secondary detection):

• Omit primary antibody to detect non-specific binding of secondary reagents

• Use normal serum from the same species as the primary antibody as negative control

Label Controls (verify FITC signal authenticity):

• Include unlabeled control sample to establish baseline autofluorescence

• Use non-specific FITC-conjugated antibody of the same isotype to determine background

• If possible, employ spectral unmixing to distinguish FITC signal from autofluorescence

How can I validate the specificity of a GUCD1 antibody in my experimental system?

Comprehensive validation requires a multi-faceted approach:

Genetic Approaches:

• CRISPR/Cas9 knockout of GUCD1 provides gold standard negative control

• siRNA/shRNA knockdown should result in proportionally reduced signal intensity

• Overexpression of tagged GUCD1 should show colocalization with antibody staining

Biochemical Validation:

• Western blotting should reveal a single band at expected molecular weight

• Mass spectrometry analysis of immunoprecipitated material confirms target identity

• Peptide competition assays using the immunogen (recombinant Human GUCD1 protein, amino acids 1-136) should abolish specific staining

Orthogonal Methods:

• Correlation with mRNA expression using fluorescent in situ hybridization

• Comparison using multiple antibodies targeting different GUCD1 epitopes

• Correlation with GFP-tagged GUCD1 expression in transfected cells

Validation under Experimental Conditions:

• Test specificity under identical fixation and preparation methods as your experiment

• Validate in the specific cell/tissue type used in your research

• Assess potential cross-reactivity with closely related proteins

What are the best methods for quantifying fluorescence intensity when using FITC-conjugated GUCD1 antibody?

Accurate quantification depends on rigorous methodology:

Microscopy-Based Quantification:

• Use confocal microscopy with standardized laser power, gain, and offset settings

• Avoid saturated pixels by checking histogram during image acquisition

• Collect Z-stack images to capture full signal distribution in 3D samples

• Maintain identical exposure settings across all experimental conditions

• Include fluorescence intensity standards in each imaging session

Image Analysis Approaches:

• Define regions of interest (ROIs) based on morphological markers

• Subtract local background from each measurement

• Measure integrated density for total protein assessment

• For colocalization studies, apply Pearson's or Mander's coefficients

• Consider automated analysis pipelines (CellProfiler, ImageJ/Fiji) for unbiased quantification

Flow Cytometry Quantification:

• Use appropriate voltage settings to place negative populations in first decade

• Include unstained, single-stained, and FMO (fluorescence minus one) controls

• Apply compensation when performing multicolor analysis with FITC (emission: 535 nm)

• Gate on viable, single cells to eliminate debris and aggregates

How do fixation methods affect the performance of FITC-conjugated GUCD1 antibody?

Fixation significantly impacts both epitope accessibility and fluorophore performance:

Optimal fixation conditions should be empirically determined for each experimental system, considering GUCD1 localization and required spatial resolution .

What approaches can be used to optimize signal-to-noise ratio when using FITC-conjugated GUCD1 antibody?

Enhancing signal-to-noise ratio involves multiple strategies:

Sample Preparation Optimization:

• Use fresh samples to minimize autofluorescence

• Optimize fixation protocol (typically brief 4% PFA fixation)

• Ensure thorough permeabilization (0.1-0.3% Triton X-100)

• Implement extensive washing with PBS containing 0.05-0.1% Tween-20

Blocking Strategies:

• Use 5-10% normal serum from secondary antibody species

• Add 1% BSA to reduce non-specific protein interactions

• Include 0.1-0.3% Triton X-100 in blocking buffer

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

Antibody Dilution Optimization:

• Perform titration experiments to determine optimal concentration

• Test dilutions ranging from 1:50 to 1:1000

• Incubate primary antibody overnight at 4°C for improved specificity

Fluorescence Enhancement:

• Use anti-fade mounting media specifically formulated for FITC

• Apply spectral unmixing to separate FITC signal (emission: 535 nm) from autofluorescence

• Use appropriate excitation (488 nm) to maximize signal-to-noise ratio

How can I troubleshoot non-specific binding when using FITC-conjugated GUCD1 antibody?

Systematic troubleshooting approach:

Identify Non-Specific Binding Sources:

• Run parallel negative controls (omitting primary antibody)

• Test in cells known to lack GUCD1 expression

• Perform peptide competition assays to distinguish specific from non-specific signals

• Evaluate blocking reagents systematically

Optimization Strategies:

• Increase blocking stringency (longer times, higher concentrations)

• Add 0.1-0.5% non-ionic detergents to reduce hydrophobic interactions

• Increase salt concentration in wash buffers (150-500 mM NaCl)

• Pre-adsorb antibody with acetone powder from non-relevant tissues

• Implement more extensive washing steps (5-6 washes of 10 minutes each)

Address Fc Receptor Binding:

• Add 10-20% serum from host species of your cells/tissue

• Use commercial Fc receptor blocking reagents

• Consider F(ab')2 fragments instead of whole IgG antibodies

Reduce Endogenous Fluorescence:

• Treat samples with sodium borohydride to reduce aldehyde-induced autofluorescence

• Use Sudan Black B to quench lipofuscin autofluorescence

• Apply copper sulfate treatment for tissue autofluorescence

What are the considerations for using FITC-conjugated GUCD1 antibody in multiplexing experiments?

Successful multiplexing requires careful planning:

Spectral Compatibility:

• FITC specifications: excitation peak ~488 nm; emission peak ~535 nm

• Avoid fluorophores with significant spectral overlap (e.g., GFP, Alexa Fluor 488)

• Compatible partners include:

  • DAPI/Hoechst (Ex: 350-405 nm, Em: 450-461 nm)

  • Rhodamine/TRITC (Ex: 532-555 nm, Em: 570-590 nm)

  • Cy5/Alexa Fluor 647 (Ex: 633-647 nm, Em: 665-670 nm)

• Perform single-color controls to establish spectral bleed-through

Staining Strategy:

• Sequential staining reduces cross-reactivity between antibodies

• Consider species origin for primary antibodies

• For multiple rabbit antibodies, use direct conjugates or sequential detection

• Test for potential interaction between antibodies

Signal Balancing:

• Adjust antibody concentrations for comparable signal intensities

• Consider differential expression levels when designing experiment

• Implement linear unmixing for closely overlapping fluorophores

Technical Considerations:

• FITC has relatively rapid photobleaching compared to other fluorophores

• Image FITC channels first in sequential imaging

• Use proper compensation controls for flow cytometry

• Consider signal amplification for low-abundance targets

How does photobleaching affect FITC-conjugated GUCD1 antibody, and what strategies can minimize this effect?

Managing photobleaching is critical for accurate results:

FITC Photobleaching Characteristics:

• More susceptible to photobleaching than newer fluorophores

• Typically loses ~20% intensity after 30 seconds of continuous illumination

• Exhibits non-linear bleaching kinetics (rapid initial decay followed by slower phase)

• Environmental factors (pH, oxygen) significantly influence stability

Acquisition Strategies:

• Reduce excitation light intensity (lower laser power)

• Minimize exposure time during image acquisition

• Focus using transmitted light before switching to fluorescence

• Utilize confocal microscopy with lower pixel dwell times

• Start with the FITC channel when performing multi-channel imaging

Sample Preparation:

• Use anti-fading mounting media containing anti-oxidants

• Remove oxygen using enzymatic oxygen scavenging systems

• Maintain slightly alkaline pH (8.0-8.5) to improve FITC stability

• Store slides at 4°C in the dark

Alternatives to Consider:

What are the optimal microscopy settings for imaging FITC-conjugated GUCD1 antibody?

Optimized settings for various microscopy platforms:

Widefield Fluorescence Microscopy:

• Excitation filter: 475-495 nm bandpass

• Emission filter: 515-545 nm bandpass

• Dichroic mirror: 505 nm long-pass

• Camera settings: 12-16 bit dynamic range, moderate gain

• Exposure time: 50-500 ms (optimized to use 70-80% of dynamic range)

Confocal Microscopy:

• Excitation laser: 488 nm (argon or solid-state)

• Emission collection: 510-550 nm

• Pinhole setting: 1 Airy unit for optimal resolution/signal

• Pixel size: 80-120 nm for optimal Nyquist sampling

• Line/frame averaging: 2-4× to improve signal-to-noise ratio

• Laser power: 1-5% of maximum (titrate to minimize photobleaching)

Multi-Channel Imaging:

• Sequential scanning to prevent bleed-through

• Acquire FITC channel first to minimize photobleaching effects

• Balance laser powers across channels for comparable signal intensity

• Implement spectral unmixing for overlapping fluorophores

Image Processing Considerations:

• Apply flat-field correction for illumination non-uniformity

• Use deconvolution to improve signal-to-noise ratio

• Apply background subtraction based on negative control samples

How can I determine the appropriate concentration of FITC-conjugated GUCD1 antibody for my specific application?

Systematic optimization approach:

Titration Method:

• Prepare serial dilutions (typically 1:10, 1:50, 1:100, 1:500, 1:1000)

• Use consistent sample preparation across dilutions

• Maintain identical imaging parameters for all samples

• Quantify signal-to-noise ratio for each dilution

• Plot signal-to-background ratio versus antibody concentration

• Select concentration at or slightly above the inflection point

Application-Specific Starting Points:

• Immunocytochemistry: 1:50-1:200 dilution (5-20 μg/ml)

• Flow cytometry: 1:10-1:100 dilution (10-100 μg/ml)

• ELISA: 1:100-1:1000 dilution (1-10 μg/ml)

Experimental Design Considerations:

• Include primary antibody omission control to assess background

• Test on both positive and negative control samples

• Consider tissue/cell-specific factors affecting antibody penetration

• Evaluate specificity at each concentration

Optimization Metrics:

• Calculate signal-to-noise ratio = (specific signal - background) / standard deviation of background

• Assess coefficient of variation across replicates

• Evaluate specificity via colocalization with orthogonal markers

What methods can be used to enhance the signal from FITC-conjugated GUCD1 antibody in tissues with high autofluorescence?

Strategies to overcome tissue autofluorescence challenges:

Pre-treatment Methods:

• Sudan Black B (0.1-0.3% in 70% ethanol) for lipofuscin autofluorescence

• Sodium borohydride (0.1-1% in PBS) for aldehyde-induced fluorescence

• Copper sulfate treatment in ammonium acetate buffer

• Photobleaching tissue sections prior to antibody application

• Commercial autofluorescence quenchers (e.g., TrueBlack®)

Signal Amplification Approaches:

• Tyramide signal amplification (TSA) to enhance FITC signal (10-100× amplification)

• Avidin-biotin amplification with FITC-conjugated streptavidin

• Multi-layer detection using anti-FITC antibodies conjugated to brighter fluorophores

• Polymer-based detection systems with multiple fluorophore molecules

Advanced Microscopy Techniques:

• Spectral imaging with linear unmixing to separate FITC from autofluorescence

• Time-gated detection to exploit different fluorescence lifetimes

• Two-photon excitation to reduce out-of-focus excitation

• Structured illumination for improved signal-to-noise ratio

Alternative Considerations:

• Replace FITC with spectrally similar but brighter fluorophores

• Shift to longer wavelength fluorophores to avoid autofluorescence spectrum

• Consider quantum dots for higher brightness and photostability

Validation Strategy:

• Include tissue-specific negative controls processed identically

• Perform parallel detection with non-fluorescent methods (e.g., chromogenic IHC)

• Validate key findings with orthogonal techniques (Western blot, qPCR)