CASTOR3 Antibody, FITC conjugated

What is CASTOR3 protein and what are its known functions?

CASTOR3, also known as Putative protein GATS (STAG3 opposite strand transcript protein), is a human protein with a molecular weight of approximately 17.8 kilodaltons. The protein is encoded by the CASTOR3 gene, which may also be referred to as GATS or STAG3OS . Current research suggests it belongs to the CASTOR family of proteins, though its precise biological functions remain under investigation. The recombinant form used for antibody generation typically encompasses amino acids 1-163 of the full protein .

What are the key properties of CASTOR3 Antibody, FITC conjugated?

CASTOR3 Antibody, FITC conjugated is a polyclonal antibody raised in rabbits against recombinant Human Putative protein CASTOR3 (amino acids 1-163) . The antibody demonstrates specificity for human CASTOR3 protein and is supplied in liquid form. The preparation is purified using Protein G chromatography (>95% purity) and is provided in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . The antibody is conjugated to the fluorescent dye FITC (fluorescein isothiocyanate), which enables direct visualization in applications like ELISA and potentially flow cytometry or fluorescence microscopy .

How does FITC conjugation work and what are its spectral properties?

FITC (fluorescein isothiocyanate) is one of the most commonly used fluorescent dyes for antibody labeling, particularly in flow cytometry analysis . The conjugation process involves the covalent attachment of FITC molecules to the antibody via primary amines (typically lysine residues) . The optimal conjugation typically results in 3-6 FITC molecules per antibody, as higher levels can lead to solubility problems and internal quenching that reduces brightness .

FITC has the following spectral characteristics:

• Excitation maximum: Typically excited by the 488 nm line of an argon laser

• Emission maximum: Approximately 530 nm

These properties make FITC-conjugated antibodies compatible with standard fluorescence microscopes and flow cytometers equipped with 488 nm lasers and appropriate emission filters.

What are the optimal storage conditions for CASTOR3 Antibody, FITC conjugated?

For maximum stability and retention of activity, CASTOR3 Antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt . It's important to avoid repeated freeze-thaw cycles as these can damage the antibody and reduce its effectiveness . Consider the following storage recommendations:

• Aliquot the antibody into smaller volumes based on experimental needs to minimize freeze-thaw cycles

• Store in light-protected containers (e.g., amber tubes or wrapped in foil) as FITC is susceptible to photobleaching

• Keep the antibody in its original storage buffer containing 50% glycerol, which helps prevent freezing damage

• When removing from storage, thaw quickly at room temperature and return unused portion to freezer promptly

What controls should be included when using CASTOR3 Antibody, FITC conjugated?

When designing experiments with CASTOR3 Antibody, FITC conjugated, the following controls should be included to ensure valid and interpretable results:

• Isotype control: Use a rabbit IgG-FITC control at the same concentration as the CASTOR3 antibody to assess non-specific binding

• Negative cell/tissue control: Include samples known not to express CASTOR3 protein

• Positive cell/tissue control: Include samples with confirmed CASTOR3 expression

• Unstained control: For flow cytometry applications, include samples with no antibody to establish autofluorescence baseline

• Blocking control: Pre-incubate the antibody with recombinant CASTOR3 protein (competitive inhibition) to confirm specificity

• Secondary-only control: If using additional detection systems, include samples treated only with secondary reagents

These controls help distinguish specific signal from background and validate the specificity of the observed staining patterns.

How can I optimize the signal-to-noise ratio when using CASTOR3 Antibody, FITC conjugated?

To maximize specific signal while minimizing background noise:

• Titrate the antibody: Determine the optimal concentration by testing a range of dilutions (e.g., 1:50, 1:100, 1:200) to identify the concentration that provides maximum specific signal with minimal background

• Optimize blocking: Use appropriate blocking buffers containing 1-5% BSA or serum from the same species as the secondary antibody (if applicable)

• Adjust fixation protocol: Different fixation methods can affect epitope accessibility; compare cross-linking fixatives (e.g., paraformaldehyde) with precipitating fixatives (e.g., methanol/acetone)

• Reduce autofluorescence:

  • For tissue sections: Use treatments like Sudan Black B (0.1-0.3%)

  • For cells with high flavoprotein content: Consider brief NaBH₄ treatment

  • For formalin-fixed samples: Incubate with 0.1-1% sodium borohydride

• Optimize permeabilization: Test different detergents (e.g., 0.1-0.5% Triton X-100, 0.1% Saponin) and incubation times

• Include antifade reagents: When mounting samples for microscopy, use mounting media with anti-photobleaching agents

What are the considerations for multiplexing CASTOR3 Antibody, FITC conjugated with other fluorophores?

When designing multi-color experiments including CASTOR3 Antibody, FITC conjugated:

• Spectral compatibility: Choose fluorophores with minimal spectral overlap with FITC (excitation: 488 nm, emission: 530 nm) . Good companions include:

  • Red-emitting dyes (e.g., PE-Cy5, APC)

  • Far-red dyes (e.g., APC-Cy7)

  • UV-excited dyes (e.g., DAPI for nuclear counterstaining)

• Signal intensity balancing: Adjust the concentration of each antibody to achieve comparable signal intensities, as FITC may be less bright than some newer fluorophores

• Compensation controls: For flow cytometry, prepare single-stained controls for each fluorophore to enable accurate compensation

• Sequential detection: For microscopy of spectrally similar fluorophores, consider sequential rather than simultaneous acquisition

• Cross-reactivity testing: Ensure antibodies from different species or isotypes are used to avoid cross-reactivity between detection systems

What is the recommended protocol for using CASTOR3 Antibody, FITC conjugated in immunofluorescence?

Based on standard protocols for FITC-conjugated antibodies and the manufacturer's recommendations for CASTOR3 Antibody:

• Sample preparation:

  • For adherent cells: Grow on glass coverslips or chamber slides

  • For tissue sections: Use freshly cut 5-10 μm sections

• Fixation and permeabilization:

  • Fix with 4% paraformaldehyde (10 min, RT) or 100% methanol (-20°C, 10 min)

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS (5-10 min, RT)

• Blocking:

  • Block with 5% normal serum or 3% BSA in PBS (1 hour, RT)

• Antibody incubation:

  • Dilute CASTOR3 Antibody, FITC conjugated at 1:50-1:200 in blocking buffer

  • Incubate 1-2 hours at room temperature or overnight at 4°C in a humidified chamber

  • Protect from light during and after antibody incubation

• Washing:

  • Wash 3× with PBS (5 min each)

• Counterstaining:

  • Optional: Counterstain nuclei with DAPI (1 μg/ml, 5 min)

  • Wash 1× with PBS

• Mounting:

  • Mount with antifade mounting medium

  • Seal edges with nail polish for long-term storage

• Imaging:

  • Visualize using a fluorescence microscope with appropriate filter sets (FITC: excitation ~488 nm, emission ~520 nm)

  • Capture images using consistent exposure settings between samples and controls

How can I optimize CASTOR3 Antibody, FITC conjugated for flow cytometry?

Flow cytometry optimization for CASTOR3 detection requires attention to several parameters:

• Cell preparation:

  • Generate single-cell suspensions with minimal debris

  • Maintain cell viability >90% for optimal results

  • For intracellular targets, use gentle fixation (2% paraformaldehyde) and permeabilization (0.1% saponin)

• Antibody titration:

  • Test serial dilutions (typically 1:25, 1:50, 1:100, 1:200) to determine optimal staining concentration

  • Evaluate signal-to-noise ratio for each concentration

  • Select the concentration with maximum separation between positive and negative populations

• Instrument settings:

  • Optimize PMT voltages using unstained cells and single-stained controls

  • Set compensation if performing multicolor analysis

  • Use logarithmic scale for fluorescence detection

• Gating strategy:

  • Gate on intact cells using FSC/SSC

  • Exclude doublets using FSC-H vs FSC-A

  • Use viability dye to exclude dead cells

  • Compare to isotype control for setting positive/negative thresholds

• Data acquisition:

  • Collect sufficient events (minimum 10,000, ideally 30,000-50,000) for statistical significance

  • Monitor fluorescence stability over time during acquisition

• Analysis considerations:

  • Quantify results as percent positive cells and/or median fluorescence intensity (MFI)

  • Use appropriate statistical tests for comparing experimental groups

What methods can be used to validate the specificity of CASTOR3 Antibody, FITC conjugated?

To confirm the specificity of CASTOR3 Antibody staining, consider implementing multiple validation approaches:

• Western blot correlation:

  • Confirm antibody detects a protein of the expected molecular weight (~17.8 kDa)

  • Compare staining pattern with an alternative CASTOR3 antibody recognizing a different epitope

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide (recombinant CASTOR3 protein, 1-163AA)

  • Observe elimination or significant reduction of signal

• Genetic validation:

  • Test staining in CASTOR3 knockout or knockdown models

  • Compare with wildtype or negative control knockdowns

• Expression correlation:

  • Compare protein detection with mRNA expression data

  • Test multiple cell lines with varying CASTOR3 expression levels

• Cross-reactivity assessment:

  • Test the antibody on samples from non-target species

  • Confirm absence of signal in tissues known not to express CASTOR3

• Orthogonal method comparison:

  • Compare results with alternative detection methods (e.g., mass spectrometry)

  • Correlate with results from in situ hybridization for CASTOR3 mRNA

How should I analyze flow cytometry data obtained with CASTOR3 Antibody, FITC conjugated?

Analysis of flow cytometry data for CASTOR3 expression should follow these guidelines:

• Quality control checks:

  • Ensure stable flow rate during acquisition (consistent events/second)

  • Check for shifts in baseline fluorescence over time

  • Verify compensation accuracy if performing multicolor analysis

• Gating strategy implementation:

  • Apply sequential gating:

    • FSC/SSC to identify cells

    • FSC-H/FSC-A to exclude doublets

    • Viability marker to exclude dead cells

    • FITC fluorescence to identify CASTOR3-positive populations

• Quantitative measurements:

  • Percentage of positive cells (above isotype control threshold)

  • Median fluorescence intensity (MFI) for relative expression level quantification

  • For heterogeneous expression, consider additional metrics such as coefficient of variation

• Visualization approaches:

  • Histogram overlays comparing experimental groups

  • Contour or density plots for bivariate analysis

  • Box plots or violin plots for statistical comparisons of MFI

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design:

    • t-test or Mann-Whitney U test for two-group comparisons

    • ANOVA or Kruskal-Wallis for multiple group comparisons

    • Consider paired tests for before/after treatments

  • Calculate effect sizes in addition to p-values

How can I distinguish between specific CASTOR3 staining and autofluorescence?

Distinguishing specific FITC signal from autofluorescence requires several strategies:

• Control-based thresholding:

  • Set positive/negative thresholds based on isotype control

  • Use fluorescence minus one (FMO) controls for multicolor experiments

• Autofluorescence characterization:

  • Analyze unstained samples in multiple channels

  • Identify cells with high autofluorescence (often phagocytes or cells with high metabolic activity)

• Multi-parameter analysis:

  • Gate on marker combinations that define your population of interest

  • Use additional surface markers to identify specific cell types

• Spectral unmixing:

  • If available, use spectral flow cytometry to mathematically separate fluorophore signals from autofluorescence

• Signal enhancement approaches:

  • For microscopy, use time-gated detection to separate FITC signal from short-lived autofluorescence

  • Consider signal amplification methods for weak CASTOR3 expression

What are the best practices for quantifying CASTOR3 expression in imaging applications?

For accurate quantification of CASTOR3 expression in fluorescence microscopy:

• Standardized image acquisition:

  • Use consistent exposure settings across all samples

  • Apply flat-field correction to compensate for uneven illumination

  • Capture multiple fields per sample (minimum 5-10) for representative analysis

• Background correction methods:

  • Subtract average intensity from regions without cells

  • Use rolling ball algorithm for uneven background

• Quantification approaches:

  • Mean fluorescence intensity within regions of interest

  • Integrated density (area × mean intensity)

  • Colocalization analysis if examining subcellular distribution

• Normalization strategies:

  • Normalize to nuclear staining for per-cell analysis

  • Use reference standards imaged under identical conditions

• Analysis software options:

  • ImageJ/FIJI with appropriate plugins

  • CellProfiler for automated high-throughput analysis

  • Commercial packages with advanced segmentation capabilities

• Data presentation:

  • Include representative images alongside quantification

  • Present data as scatter plots showing individual measurements, not just means