STATH Antibody, FITC conjugated

What is STATH Antibody, FITC conjugated, and what are its spectral properties?

STATH Antibody, FITC conjugated consists of an antibody against statherin (a salivary protein) that has been chemically linked to fluorescein isothiocyanate (FITC). FITC is a fluorochrome dye that absorbs ultraviolet or blue light and emits yellow-green fluorescence. The excitation and emission peak wavelengths for FITC are approximately 495nm and 525nm, respectively. This conjugation enables direct visualization of statherin protein in various biological samples without requiring secondary antibody staining . The fluorescence signal disappears when the excitation light source is removed, making it ideal for controlled visualization experiments. The conjugation process is typically performed in a manner that preserves the biological activity and binding specificity of the antibody .

What are the recommended storage conditions for STATH Antibody, FITC conjugated?

STATH Antibody, FITC conjugated should be stored at -20°C, similar to other FITC-conjugated antibodies. The antibody is typically supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol to maintain stability . To prevent degradation from repeated freeze-thaw cycles, it is strongly recommended to aliquot the antibody into multiple small volumes before freezing . When handling the antibody, minimize exposure to light as FITC is susceptible to photobleaching. For short-term storage (less than one week), the antibody can be kept at 4°C in the dark, but long-term storage requires freezing to preserve fluorescence intensity and antibody activity.

What applications is STATH Antibody, FITC conjugated suitable for?

STATH Antibody, FITC conjugated can be utilized in multiple research applications including:

• Western Blotting (WB): For detecting denatured statherin protein in complex samples with typical dilutions ranging from 1:300-1:5000 .

• Immunofluorescence on paraffin-embedded tissues (IF/IHC-P): For visualizing statherin distribution in fixed tissue sections at dilutions of 1:50-1:200 .

• Immunocytochemistry (IF/ICC): For studying subcellular localization of statherin in cultured cells at dilutions of 1:50-1:200 .

• Flow Cytometry: For quantitative analysis of statherin expression in cell populations, allowing for simultaneous measurement of multiple parameters .

• Fluorescence Microscopy: For direct visualization of statherin in fixed or live cells without requiring secondary antibody detection steps.

Each application requires specific optimization of antibody concentration, incubation times, and buffer compositions to achieve optimal signal-to-noise ratios.

How do I determine the optimal concentration of STATH Antibody, FITC conjugated for my experiment?

Determining the optimal concentration of STATH Antibody, FITC conjugated requires titration experiments for each specific application and sample type. Begin with the manufacturer's recommended dilution ranges (1:50-1:200 for immunofluorescence, 1:300-1:5000 for Western blotting) . Prepare a series of antibody dilutions (e.g., 1:25, 1:50, 1:100, 1:200, 1:400) and test them on your samples using identical conditions. The optimal concentration provides maximum specific signal with minimal background. For flow cytometry, analyze the separation index or staining index at each concentration by comparing the median fluorescence intensity of positive versus negative populations. For microscopy applications, evaluate signal intensity, specificity, and background levels. Document your optimization process systematically, as different sample types (tissue sections vs. cell lines) often require different optimal concentrations.

What controls should I include when working with STATH Antibody, FITC conjugated?

Proper controls are essential for reliable interpretation of experiments using STATH Antibody, FITC conjugated:

• Negative Controls:

  • Isotype control: A FITC-conjugated immunoglobulin of the same isotype and host species but lacking specificity for the target

  • Secondary antibody-only control (for indirect detection methods)

  • Unstained samples to establish autofluorescence baseline

• Positive Controls:

  • Samples known to express statherin protein (e.g., salivary gland tissue)

  • Recombinant statherin protein or overexpression systems

• Specificity Controls:

  • Pre-absorption with purified antigen to confirm specificity

  • siRNA knockdown or CRISPR knockout of STATH gene

  • Testing in multiple cell lines with varying STATH expression levels

• Technical Controls:

  • Fluorescence minus one (FMO) controls for multicolor flow cytometry

  • Anti-FITC antibody to verify successful conjugation and FITC activity

These controls help distinguish specific signals from artifacts and allow for proper interpretation of experimental results.

How can I optimize STATH Antibody, FITC conjugated staining for multi-parameter flow cytometry?

Optimizing STATH Antibody, FITC conjugated for multi-parameter flow cytometry requires strategic panel design and protocol refinement:

• Panel Design Considerations:

  • Since FITC emits in the 525nm range, avoid fluorophores with significant spectral overlap (e.g., PE, GFP)

  • Place FITC on high-abundance targets when possible, as it has moderate brightness

  • Implement proper compensation controls for each fluorophore in your panel

  • Use fluorophores with distinct emission spectra for other markers

• Protocol Optimization:

  • Titrate antibody concentration specifically for flow cytometry to determine optimal separation

  • Assess the quenching effect using anti-FITC antibodies at 1:100 dilution to confirm specificity

  • Use buffered solutions containing protein (1% BSA) to reduce non-specific binding

  • Include viability dyes to exclude dead cells, which often exhibit autofluorescence

  • If detecting intracellular statherin, ensure optimal fixation and permeabilization conditions

• Instrument Setup:

  • Use single-stained controls for proper compensation

  • Adjust PMT voltages to properly visualize both negative and positive populations

  • Collect sufficient events (10,000+) for reliable statistical analysis

  • Consider standardization beads for day-to-day consistency

A systematic approach comparing cell types with known statherin expression levels will help establish reliable gating strategies for identifying true positive populations.

What are the methods to validate the specificity of STATH Antibody, FITC conjugated in different tissue types?

Validating the specificity of STATH Antibody, FITC conjugated across different tissue types requires multiple complementary approaches:

• Western Blot Correlation:

  • Perform Western blot analysis of tissue lysates using the unconjugated version of the same STATH antibody

  • Compare band patterns and intensities across tissues to microscopy results

  • Load varying amounts of sample (0.5-10 μg) to establish detection limits

• Peptide Competition Assays:

  • Pre-incubate the antibody with purified statherin peptide prior to staining

  • Monitor reduction in signal intensity as confirmation of specificity

  • Include graded concentrations of blocking peptide to demonstrate dose-dependent inhibition

• Cross-Validation with Alternative Detection Methods:

  • Compare FITC-conjugated antibody staining patterns with those obtained using:

    • Different STATH antibody clones

    • In situ hybridization for STATH mRNA

    • Mass spectrometry data on tissue proteomes

• Genetic Validation:

  • Test antibody in tissues from STATH knockout models or following STATH knockdown

  • Compare staining between tissues with naturally varying STATH expression levels

  • Perform staining on transfected cells with controlled STATH expression

• Multiple Tissue Analysis:

  • Create a tissue microarray containing multiple tissue types

  • Quantify staining intensities and patterns across tissues

  • Compare results with published STATH expression databases

Documentation of these validation steps significantly increases confidence in the specificity of staining patterns observed with STATH Antibody, FITC conjugated.

How can potential photobleaching of FITC affect STATH localization studies, and what strategies can minimize this effect?

Photobleaching of FITC conjugated to STATH antibody can significantly impact localization studies:

Effects of Photobleaching:

• Decreased signal intensity over time, potentially obscuring true localization patterns

• Uneven bleaching across the sample leading to artifacts in distribution analysis

• Challenges in time-lapse imaging and co-localization studies

• Difficulty in quantitative comparisons between samples with different exposure histories

Strategies to Minimize Photobleaching:

• Sample Preparation and Imaging Setup:

  • Use anti-fade mounting media containing radical scavengers

  • Store slides in the dark and minimize exposure during handling

  • Optimize microscope settings to use minimal excitation intensity while maintaining adequate signal

  • Consider using confocal microscopy with precisely controlled laser power

• Image Acquisition Approaches:

  • Acquire reference images at low magnification before detailed high-power imaging

  • Use neutral density filters to reduce excitation intensity

  • Implement shorter exposure times with signal averaging

  • Utilize computational approaches like deconvolution to enhance signal from lower-exposure images

• Alternative Methodologies:

  • Consider using more photostable fluorophores (like Alexa Fluor dyes) if available

  • Employ alternative detection methods for validation (enzyme-linked detection systems)

  • Use photobleaching correction algorithms during image analysis

  • Implement spin-scanning confocal microscopy for reduced photobleaching

• Quantification Considerations:

  • Always image control and experimental samples under identical conditions

  • Include reference standards for normalization

  • Account for bleaching rates in quantitative analyses

  • Consider photobleaching correction in time-lapse experiments

By implementing these strategies, researchers can obtain more reliable data on STATH localization while minimizing artifacts from FITC photobleaching.

How can I troubleshoot high background when using STATH Antibody, FITC conjugated in immunofluorescence?

High background is a common challenge when using FITC-conjugated antibodies. Here are systematic troubleshooting approaches:

Common Causes and Solutions:

Systematic Approach to Background Reduction:

• Start with proper controls to identify the nature of background (non-specific binding vs. autofluorescence)

• Implement one change at a time and document effects

• Consider dual approaches: reduce background while enhancing specific signal

• Validate improvements across multiple samples

What protocols can be used to combine STATH Antibody, FITC conjugated with other fluorophore-labeled antibodies for co-localization studies?

Co-localization studies with STATH Antibody, FITC conjugated require careful planning to avoid spectral overlap and ensure compatible protocols:

Recommended Protocol for Multi-color Immunofluorescence:

• Panel Design:

  • Choose fluorophores with minimal spectral overlap with FITC (495/525nm)

  • Recommended combinations: FITC (green) + TRITC/Texas Red (red) + DAPI (blue)

  • Avoid PE, GFP, or other green fluorophores that would interfere with FITC detection

• Sample Preparation:

  • Fix samples using 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.1-0.3% Triton X-100 in PBS (5-10 minutes)

  • Block with 5% normal serum (from species unrelated to antibody hosts) + 1% BSA in PBS (1-2 hours)

• Primary Antibody Incubation Options:

  • Sequential Approach: Incubate with unconjugated antibody first, followed by its secondary antibody, then STATH Antibody, FITC conjugated

  • Simultaneous Approach: If antibodies are from different host species, incubate with all primary antibodies together (including STATH Antibody, FITC conjugated) overnight at 4°C

• Controls for Co-localization:

  • Single-color controls to establish bleed-through parameters

  • Secondary antibody-only controls to assess non-specific binding

  • Biological controls (known co-localizing or non-co-localizing proteins)

• Advanced Imaging Considerations:

  • Use sequential scanning on confocal microscopes to minimize crosstalk

  • Implement appropriate chromatic aberration corrections

  • Consider structured illumination or super-resolution techniques for detailed co-localization analysis

  • Quantify co-localization using Pearson's or Mander's coefficients

This approach allows for reliable investigation of protein interactions or spatial relationships between statherin and other proteins of interest.

How can STATH Antibody, FITC conjugated be used in studying calcium regulation in salivary function?

STATH Antibody, FITC conjugated provides valuable tools for investigating statherin's role in calcium regulation within salivary function:

Experimental Approaches:

• Temporal Expression Studies:

  • Use STATH Antibody, FITC conjugated (1:50-1:200 dilution) to track statherin expression changes in salivary gland tissue during:

    • Development stages

    • Stimulated vs. resting states

    • Pathological conditions (e.g., Sjögren's syndrome)

  • Correlate statherin localization with calcium-binding proteins using multi-color immunofluorescence

• Calcium Precipitation Assays:

  • Visualize statherin localization at calcium phosphate nucleation sites

  • Combine with calcium indicators (e.g., Alizarin Red) to correlate statherin presence with mineralization

  • Track changes in statherin distribution during experimental manipulation of calcium levels

• In Vitro Functional Studies:

  • Use STATH Antibody, FITC conjugated in flow cytometry (1:100) to sort cell populations based on statherin expression

  • Compare calcium handling between statherin-positive and negative populations

  • Combine with calcium-sensing fluorescent dyes to simultaneously monitor calcium flux and statherin localization

• Tooth Surface Studies:

  • Examine statherin adsorption to hydroxyapatite in experimental systems

  • Visualize the pellicle formation process and statherin's role in mineral homeostasis

  • Correlate statherin binding with calcium ion concentration at tooth surfaces

• Methodological Protocol:

  • Fixation: 4% paraformaldehyde (preserves calcium distribution)

  • Blocking: 5% BSA in TBS (pH 7.4) for 1 hour

  • Primary staining: STATH Antibody, FITC conjugated (1:100) overnight at 4°C

  • Calcium staining: Add calcium-specific dyes in the final steps

  • Mounting: Use anti-fade media with DAPI for nuclear counterstain

These approaches provide comprehensive insights into statherin's functional role in calcium regulation within salivary physiology.

What are the considerations when using STATH Antibody, FITC conjugated in different model organisms?

Using STATH Antibody, FITC conjugated across different model organisms requires careful consideration of several factors:

Cross-Species Reactivity Considerations:

Protocol Adaptations for Different Model Systems:

• Tissue-specific considerations:

  • Salivary tissue: May contain endogenous fluorescent compounds requiring additional blocking

  • Tooth/enamel: Decalcification protocols must be compatible with epitope preservation

  • Cell cultures: Different fixation requirements based on cell type

• Modified validation approaches:

  • Western blot verification at expected molecular weight for the specific species

  • Include known positive control tissues from the target species

  • Consider peptide competition assays using species-specific protein sequences

• Technical adaptations:

  • Optimize antigen retrieval methods specific to each species' tissue composition

  • Adjust permeabilization conditions based on tissue density and fixation method

  • Consider tyramide signal amplification for low expression systems

• Alternative detection strategies:

  • For challenging tissues, consider using unconjugated STATH antibody with species-appropriate secondary antibodies

  • Implement alternative visualization methods like enzyme-based detection systems

By addressing these species-specific considerations, researchers can successfully apply STATH Antibody, FITC conjugated across diverse model organisms while maintaining experimental rigor.

How does the molar ratio of FITC to antibody affect the performance of STATH Antibody, FITC conjugated?

The molar ratio of FITC to antibody (F/P ratio) significantly impacts the performance characteristics of STATH Antibody, FITC conjugated:

Impact of Different F/P Ratios:

Methods to Determine F/P Ratio:

The F/P ratio can be calculated using spectrophotometric measurements according to the formula:
$\text{F/P ratio} = \frac{A_{495} \times \text{MW of antibody}}{195,000 \times A_{280} - (0.35 \times A_{495})}$

Where:

• A₄₉₅ is the absorbance at 495 nm

• A₂₈₀ is the absorbance at 280 nm

• MW of antibody is typically 150,000 Da for IgG

• 195,000 is the molar extinction coefficient of IgG at 280 nm

• 0.35 accounts for FITC absorption at 280 nm

Optimization Strategies:

• For new research applications, test multiple F/P ratio preparations:

  • Small-scale FITC conjugations using three different molar ratios

  • Evaluate each preparation for specificity and sensitivity in your experimental system

• For established protocols with known target expression levels:

  • High abundance targets: Use lower F/P ratio conjugates

  • Low abundance targets: Consider higher F/P ratio conjugates with appropriate controls

• When working with commercial preparations:

  • Request information on the F/P ratio from the manufacturer

  • Verify performance in your specific application regardless of reported specifications

Understanding and optimizing the F/P ratio is crucial for achieving reliable and reproducible results with STATH Antibody, FITC conjugated across different experimental platforms.