FAU Antibody, FITC conjugated

What is FAU and what does a FAU antibody detect?

FAU (Finkel-Biskis-Reilly Murine Sarcoma Virus Ubiquitously Expressed) is a protein initially identified in the FBR murine sarcoma virus. FAU antibodies can target different regions of the protein, with available options including antibodies recognizing the C-terminal region or N-terminal region of human FUBI (FAU ubiquitin-like protein) . These antibodies are typically validated for reactivity with human and mouse samples, though some antibodies show broader species reactivity . Researchers should select antibodies targeting specific epitopes based on their experimental design and the structural accessibility of the target region in their particular application.

What applications are supported by FITC-conjugated antibodies?

FITC-conjugated antibodies are widely used in fluorescence-based applications including:

• Immunohistochemistry and immunocytochemistry

• Flow cytometry and cell sorting

• Immunofluorescence microscopy

• Surface labeling experiments

• Fluorophore-linked immunosorbent assays

These antibodies are particularly valuable when direct detection is preferred over secondary antibody systems, as they eliminate potential cross-reactivity issues and simplify experimental workflows . The bright green fluorescence of FITC (emission maximum at 525 nm) makes it compatible with most fluorescence detection systems and ideal for multicolor experiments when combined with fluorophores of different spectral properties .

How should I choose between different FAU antibody options?

When selecting a FAU antibody for FITC conjugation or choosing a pre-conjugated product, consider:

• Target epitope: C-terminal vs. N-terminal targeting affects accessibility in different applications

• Host species: Choose based on compatibility with other antibodies in multiplex experiments

• Clonality: Polyclonal antibodies offer broader epitope recognition while monoclonal antibodies provide higher specificity

• Validated applications: Ensure the antibody is validated for your specific application (WB, IHC, IF, flow cytometry)

• Species reactivity: Confirm cross-reactivity with your experimental model organism

Always validate antibody performance in your specific experimental system before proceeding with full-scale studies.

What controls should I include when using FAU-FITC antibodies?

Proper controls are essential for interpreting results from experiments using FAU-FITC antibodies:

• Isotype control: Use a FITC-conjugated antibody of the same isotype but irrelevant specificity

• Negative control tissues/cells: Include samples known to be negative for FAU expression

• Blocking controls: Pre-incubate with unconjugated antibody or competing peptide

• Autofluorescence control: Examine unstained samples to assess background fluorescence

• Knockout/knockdown validation: When possible, validate specificity using FAU-depleted samples

These controls help distinguish specific from non-specific signals and ensure accurate data interpretation.

How does the fluorophore-to-protein (F/P) ratio affect experimental outcomes?

The F/P ratio (molar ratio of fluorophore to protein) significantly impacts antibody performance:

Overlabeling antibodies (F/P ratios >6) typically results in:

• Increased non-specific binding and background fluorescence

• Decreased quantum yield due to self-quenching

• Potential alterations in antibody specificity and binding affinity

• Increased risk of antibody aggregation and precipitation

For optimal results, researchers should test different conjugation ratios and select the one providing the best signal-to-noise ratio for their specific application.

What are the critical factors in optimizing FITC-FAU antibody performance in flow cytometry?

Optimizing FITC-conjugated antibody performance in flow cytometry requires attention to several factors:

• Signal compensation: FITC has spectral overlap with other common fluorophores, particularly PE, requiring proper compensation

• Titration: Determine optimal antibody concentration to maximize specific signal while minimizing background

• Fixation effects: FITC fluorescence may be affected by fixatives; paraformaldehyde concentrations should typically be kept below 1%

• pH sensitivity: FITC fluorescence is optimal at pH >7.0 and diminishes in acidic conditions

• Photobleaching: Minimize light exposure during sample preparation and acquisition

• Instrument setup: Calibrate using appropriate beads to ensure consistent detection of FITC signals

When designing multiplex panels, place FITC on abundant targets or pair with bright fluorophores on less abundant targets to maximize detection sensitivity.

How can I determine if my FAU-FITC antibody maintains functional binding after conjugation?

After FITC conjugation, it's essential to verify that the antibody retains its specificity and binding properties:

• Comparative analysis: Test performance against unconjugated antibody in parallel experiments

• Antigen competition assay: Pre-incubate with purified antigen to demonstrate signal reduction

• Spectrophotometric assessment: Calculate the F/P ratio to ensure optimal labeling

• Activity assays: Compare binding curves of conjugated versus unconjugated antibody

• Western blot validation: Confirm detection of the expected molecular weight protein band (~28-33 kDa for FAU/FUBI)

For FAU antibodies specifically, validation can include detection of the 28-33 kDa band in western blot or appropriate cellular localization patterns in immunofluorescence microscopy .

What methodological adaptations are needed when using FAU-FITC antibodies for different specimen types?

Different specimens require specific methodological considerations:

Fixed tissues:

• Optimize fixative type and concentration to preserve both antigen structure and fluorescence intensity

• Consider antigen retrieval methods if necessary for FAU detection

• Extend incubation times to allow adequate tissue penetration

• Include lipofuscin quenching steps for tissues with high autofluorescence

Cell suspensions:

• Use appropriate permeabilization methods if detecting intracellular FAU epitopes

• Optimize antibody concentration specifically for flow applications (typically lower than for microscopy)

• Include viability dyes to exclude dead cells with potentially non-specific binding

Live cells:

• Verify antibody performance under non-fixed conditions

• Minimize exposure time and light intensity to prevent phototoxicity

• Maintain physiological temperature and pH during imaging

What is the optimal protocol for conjugating FITC to FAU antibodies?

For optimal FITC conjugation to FAU antibodies, follow these methodological guidelines:

• Buffer preparation: Dissolve one sodium carbonate-bicarbonate capsule in 50 ml deionized water to create 0.1 M carbonate-bicarbonate buffer (pH 9.0)

• Antibody preparation: Prepare FAU antibody at 5.0 mg/ml in the carbonate-bicarbonate buffer

• FITC reaction:

  • For small-scale optimization, test three different FITC:antibody molar ratios (5:1, 10:1, 20:1)

  • Reconstitute FITC in carbonate-bicarbonate buffer

  • Add appropriate volume of FITC solution to antibody

  • Incubate for 2 hours at room temperature in darkness

• Purification: Remove unreacted FITC using gel filtration through a column equilibrated with PBS

• Quality control: Determine the F/P ratio spectrophotometrically by measuring absorbance at 280 nm (protein) and 495 nm (FITC)

After identifying the optimal molar ratio in small-scale tests, scale up the procedure maintaining the same reagent concentrations and ratios.

How should FAU-FITC conjugated antibodies be stored to maintain activity?

Proper storage is critical for maintaining FITC-conjugated antibody performance:

• Temperature: Store at 4°C for short-term use (up to 1 month); for long-term storage, aliquot and store at -20°C

• Light protection: Always protect from light to prevent photobleaching of the FITC fluorophore

• Aliquoting: Prepare single-use aliquots to avoid repeated freeze-thaw cycles

• Stabilizers: Addition of 1% BSA and 0.1% sodium azide helps maintain stability

• Carrier proteins: Presence of carrier proteins (e.g., BSA) reduces adsorption to container surfaces

• pH conditions: Maintain neutral to slightly basic pH conditions (7.2-7.4)

When properly stored, FITC-conjugated antibodies typically maintain activity for at least 6-12 months, though gradual loss of fluorescence intensity may occur over time.

What dilution ranges work best for different applications of FAU-FITC antibodies?

Optimal dilution ranges vary by application type:

These ranges serve as starting points; optimal dilutions should be determined empirically for each specific antibody and experimental system through titration experiments.

How can I determine the concentration and quality of my FAU-FITC conjugate?

Assessing conjugate quality involves several key measurements:

• Protein concentration determination:

  • Measure absorbance at 280 nm (A280)

  • Apply correction factor for FITC contribution to A280

  • Calculate using antibody extinction coefficient (typically 1.4 for IgG at 1 mg/ml)

• F/P ratio calculation:

  • Measure absorbance at 495 nm (A495) for FITC

  • Calculate molar F/P ratio using the formula:
    F/P = (A495 × dilution factor) / (195 × protein concentration in mg/ml)

  • Optimal F/P ratios typically range from 2-6

• Functional quality assessment:

  • Compare staining pattern with unconjugated antibody + secondary detection

  • Verify signal-to-noise ratio in actual experimental conditions

  • Confirm expected subcellular localization pattern for FAU protein

• Purity assessment:

  • Run SDS-PAGE to confirm absence of aggregates or degradation products

  • Perform size exclusion chromatography to verify homogeneity

How can I reduce high background signals when using FAU-FITC antibodies?

High background with FITC-conjugated antibodies can have multiple causes and solutions:

Causes and solutions for high background:

• Overlabeled antibody (high F/P ratio):

  • Reduce FITC:antibody ratio during conjugation

  • Dilute the existing conjugate further

  • Use conjugates with F/P ratios <6

• Non-specific binding:

  • Increase blocking with serum from the same species as the experimental samples

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Pre-adsorb antibodies against irrelevant tissues

• Tissue/cell autofluorescence:

  • Include quenching steps (e.g., sodium borohydride treatment)

  • Use Sudan Black B treatment (0.1-0.3%) to reduce lipofuscin fluorescence

  • Consider spectral unmixing during image acquisition

• Fixation artifacts:

  • Optimize fixative concentration and duration

  • Test alternative fixation methods

  • Include antigen retrieval steps if necessary

How do I distinguish between specific FAU-FITC signal and autofluorescence?

Differentiating specific signal from autofluorescence requires methodical controls:

• Unstained control: Examine samples without any antibody to establish natural autofluorescence levels

• Isotype control: Use FITC-conjugated antibody of matching isotype but irrelevant specificity to identify non-specific binding

• Spectral fingerprinting:

  • Autofluorescence typically has broader emission spectrum than FITC

  • Use spectral detectors or multiple bandwidth filters to characterize signal

  • Apply spectral unmixing algorithms during analysis

• Signal extinction test:

  • Photobleach small regions and measure recovery rate

  • Specific FITC signals and autofluorescence have different photobleaching kinetics

• Negative control samples:

  • Use tissues/cells known to be negative for FAU expression

  • Compare signal patterns between positive and negative samples

What strategies can enhance detection sensitivity for FAU using FITC conjugates?

When working with low abundance targets or weak signals, several approaches can enhance detection:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) compatible with FITC detection

  • Multi-layer approaches (biotin-streptavidin systems coupled with FITC)

  • Enzyme-mediated amplification systems

• Optical enhancement:

  • Use objectives with higher numerical aperture

  • Apply deconvolution algorithms to microscopy images

  • Employ confocal or super-resolution techniques for improved signal-to-noise ratio

• Sample preparation optimization:

  • Test different fixation protocols to maximize epitope accessibility

  • Optimize antigen retrieval methods for tissue sections

  • Adjust permeabilization conditions for intracellular targets

• Acquisition optimization:

  • Increase exposure/dwell time (balanced against photobleaching)

  • Use PMT/detector settings optimized for FITC wavelengths

  • Apply appropriate bandpass filters to capture peak FITC emission

How can I quantitatively analyze FAU-FITC signals across different experimental conditions?

Quantitative analysis of FITC signals requires standardization approaches:

• Calibration standards:

  • Use calibrated FITC beads to normalize fluorescence intensity

  • Include consistent positive controls across experiments

  • Measure molecules of equivalent soluble fluorochrome (MESF) for flow cytometry

• Image analysis methodologies:

  • Apply consistent thresholding algorithms

  • Use background subtraction methods

  • Employ region of interest (ROI) analysis with fixed parameters

• Normalization approaches:

  • Express signals relative to housekeeping protein controls

  • Calculate relative fluorescence units (RFU) using reference standards

  • Use ratiometric analysis against internal controls

• Statistical considerations:

  • Account for spectral overlap when multiple fluorophores are used

  • Apply appropriate statistical tests based on data distribution

  • Consider cell-to-cell variability in single-cell analyses

• Reporting standards:

  • Document all acquisition parameters (exposure time, gain settings)

  • Report antibody concentration, clone/catalog number, and dilution

  • Include details of instrumentation and filter sets used

Consistent methodology across experiments enables reliable comparison of FAU expression or localization between different experimental conditions or treatment groups.