FABP9 Antibody, FITC conjugated

What is FABP9 and why is it studied with fluorescent antibodies?

Fatty acid-binding protein 9 (FABP9) is a member of the intracellular lipid-binding protein family involved in fatty acid transport and metabolism. FITC-conjugated antibodies targeting FABP9 enable visualization of protein localization and quantitation in cellular contexts through fluorescence microscopy and flow cytometry. The protein is encoded by the human gene located at locus hsa:646480 according to KEGG database information . Researchers study FABP9 to understand its role in lipid transport pathways and potential involvement in various physiological and pathological processes.

What are the technical specifications of commercially available FABP9 Antibody, FITC conjugated?

FABP9 Antibody (FITC) is typically available as a Rabbit Polyclonal antibody specifically designed for human FABP9 detection. The technical specifications generally include:

These antibodies are typically stored in 0.01 M PBS (pH 7.4) with 0.03% Proclin-300 and 50% glycerol to maintain stability .

How does FITC conjugation work and what are its advantages for FABP9 research?

FITC conjugation involves the reaction between fluorescein isothiocyanate and free amino groups (primarily lysine residues) of proteins to form stable conjugates. The reaction forms thiourea bonds that covalently link the fluorophore to the antibody .

FITC offers several advantages for FABP9 research:

• High quantum efficiency providing bright fluorescence signals

• Well-established excitation/emission profile (495nm/525nm) compatible with most fluorescence microscopy systems

• Stability of FITC-protein conjugates under appropriate storage conditions

• Compatibility with multicolor imaging when combined with other fluorophores

• Extensive literature precedent providing comparative reference data

The conjugation process must be carefully controlled as overlabeling can alter specificity, cause protein aggregation, or increase non-specific binding, while high fluorophore to antibody ratios (molar F/P >6) can decrease quantum yield due to self-quenching effects .

What are the recommended storage conditions for FABP9 Antibody, FITC conjugated?

For optimal preservation of antibody function and fluorescence signal, FABP9 Antibody, FITC conjugated should be:

• Aliquoted into small volumes immediately after reconstitution to minimize freeze-thaw cycles

• Stored at -20°C for long-term preservation

• Protected from light at all times to prevent photobleaching of the FITC fluorophore

• Avoid repeated freeze/thaw cycles which can reduce antibody activity and fluorescence intensity

• For short-term storage (1-2 weeks), storage at 4°C is acceptable if protected from light

For reconstituted antibody preparations intended for extended use, addition of 1% (w/v) BSA and 0.1% (w/v) sodium azide to the antibody solution can help maintain stability during storage at 2-8°C .

How can I optimize FITC-conjugated FABP9 antibody for single-molecule tracking studies?

Single-molecule tracking (SMT) with FITC-conjugated FABP9 antibodies requires careful optimization of several parameters:

• Conjugation Optimization: The fluorophore-to-protein (F/P) ratio should be carefully controlled. For SMT applications, an F/P ratio of 2-4 typically provides the best balance between brightness and antibody functionality . Calculate the F/P ratio using the equation:

  Molar F/P = (2.77 × A495) / (A280 – (0.35 × A495))

• Fragment Generation: Consider using Fab' fragments rather than whole IgG antibodies. The bivalency of whole antibodies can cause bridging effects that alter protein behavior. Generate Fab' fragments using the following approach:

  • Digest IgG into F(ab')2 using pepsin

  • Perform limited reduction of F(ab')2 with cysteamine to generate Fab' with free sulfhydryl groups in the hinge region

  • This approach preserves the interchain disulfide bond within Fab', maintaining structural stability and binding capability

• Imaging Parameters: Use total internal reflection fluorescence microscopy (TIRFM) with appropriate laser power to balance signal strength and photobleaching. Typical settings include:

  • 488 nm laser excitation at 1-5 mW power (at the sample)

  • Exposure times of 10-100 ms

  • Electron-multiplying CCD cameras with high quantum efficiency

  • Drift correction using fiducial markers

• Background Reduction: Implement careful blocking steps with appropriate blocking buffers containing 1-5% BSA or 5-10% normal serum from the same species as the secondary antibody to minimize non-specific binding .

What approaches can be used to determine the optimal FITC-to-FABP9 antibody ratio for specific applications?

Determining the optimal FITC-to-antibody ratio requires a systematic approach that balances brightness with maintained antibody function:

• Small-Scale Optimization: Perform test conjugations at multiple FITC-to-antibody molar ratios (typically 5:1, 10:1, and 20:1) as follows:

  • Prepare three reaction mixtures with different molar ratios of FITC to antibody

  • Perform conjugation under identical buffer conditions (typically carbonate buffer, pH 9.0)

  • Purify conjugates and determine the resulting F/P ratios spectrophotometrically

  • Test each conjugate for binding specificity and signal-to-noise ratio in your specific application

• Spectrophotometric Assessment: Measure absorbance at 280 nm (protein) and 495 nm (FITC) to calculate the F/P ratio. The optimal ranges are:

  • For immunohistochemistry: F/P ratio of 2-4

  • For flow cytometry: F/P ratio of 3-6

  • For high-sensitivity microscopy: F/P ratio of 1-3

• Functional Validation: Test each conjugate batch with:

  • Positive and negative control samples

  • Titration experiments to determine optimal working concentration

  • Competition assays with unlabeled antibody to confirm specificity

• Application Optimization: Based on initial results, scale up to prepare larger batches using the optimal conjugation ratio for your specific application .

How can I distinguish between specific and non-specific binding when using FITC-conjugated FABP9 antibodies?

Discriminating between specific and non-specific binding is critical for accurate data interpretation:

• Negative Controls: Always include multiple controls:

  • Isotype control: Use FITC-conjugated non-specific IgG of the same isotype from the same host species

  • Secondary-only control: Omit primary antibody to assess background from the detection system

  • Blocking peptide competition: Pre-incubate the antibody with excess recombinant FABP9 protein (immunogen)

  • Knockout/knockdown control: Use cells with FABP9 gene knockout or knockdown

• Dilution Series: Perform a dilution series of the antibody to identify the concentration at which specific signal-to-noise ratio is optimal. Plot signal intensity versus antibody concentration to identify saturation points.

• Cross-Adsorption Techniques: If cross-reactivity is observed, use cross-adsorbed antibodies where the antibody preparation has been pre-adsorbed against potentially cross-reactive proteins.

• Spectral Analysis: Analyze emission spectra of stained samples to distinguish autofluorescence (typically broader emission spectrum) from specific FITC signal (characteristic emission peak at 525 nm).

• Photobleaching Kinetics: FITC-conjugated antibodies show characteristic photobleaching kinetics distinct from cellular autofluorescence, which can be used to distinguish specific binding .

What factors affect the stability of FITC-conjugated FABP9 antibodies and how can stability be improved?

Several factors influence the stability of FITC-conjugated antibodies:

• pH Sensitivity: FITC fluorescence is pH-dependent, with optimal fluorescence at slightly alkaline pH (7.5-8.5). To stabilize pH:

  • Use buffers with adequate buffering capacity (phosphate buffers at 0.01-0.1 M)

  • Avoid acidic conditions during storage and application

  • Monitor pH throughout experimental procedures

• Photobleaching: FITC is susceptible to photobleaching. Implement these strategies:

  • Minimize exposure to light during storage and handling

  • Use antifade reagents such as ProLong Gold or SlowFade in mounting media

  • Reduce excitation light intensity and exposure time during imaging

  • Consider including oxygen scavengers in imaging buffers

• Protein Stability: Antibody denaturation affects binding capacity. Enhance stability by:

  • Adding protein stabilizers (1% BSA) to storage buffers

  • Including glycerol (20-50%) to prevent freeze-thaw damage

  • Using preservatives such as 0.01% sodium azide to prevent microbial growth

  • Avoiding repeated freeze-thaw cycles by preparing small aliquots

• Conjugate Stabilization: The FITC-protein linkage can hydrolyze over time. Improve conjugate stability by:

  • Storing at -20°C in the dark

  • Using proper blocking buffers that do not interfere with FITC fluorescence

  • Considering alternative Fab' generation methods that maintain structural stability

How do I resolve weak signals when using FITC-conjugated FABP9 antibodies?

Weak FITC signals can stem from multiple causes with specific solutions:

• Insufficient Target Protein: If FABP9 expression is naturally low:

  • Optimize fixation methods to preserve epitope structure (try 4% paraformaldehyde for 10-15 minutes)

  • Implement antigen retrieval methods if applicable

  • Use signal amplification systems such as tyramide signal amplification

  • Consider sample concentration or enrichment techniques

• Suboptimal Antibody Concentration: Titrate the antibody to determine optimal concentration:

  • Prepare a dilution series (typically 1:50 to 1:1000)

  • Test each dilution on positive control samples

  • Select the dilution providing maximum specific signal with minimal background

• Fluorophore Quenching: To minimize FITC self-quenching:

  • Use conjugates with appropriate F/P ratios (typically 2-4)

  • Use antifade reagents in mounting media

  • Consider reducing agents in buffers to maintain fluorophore in reduced state

• Microscope Settings: Optimize imaging parameters:

  • Use appropriate excitation filters (maximum at 488-495 nm)

  • Ensure emission filters capture the FITC emission peak (515-525 nm)

  • Adjust detector gain and laser power based on sample brightness

  • Consider longer exposure times balanced against photobleaching concerns

How can I minimize background fluorescence when using FITC-conjugated antibodies in tissues with high autofluorescence?

Tissues with high autofluorescence (like liver, kidney, brain) present challenges for FITC-based detection:

• Autofluorescence Reduction Treatments:

  • Sodium borohydride treatment (1% solution for 10 minutes) to reduce aldehyde-based autofluorescence

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

  • Copper sulfate treatment (10mM CuSO₄ in 50mM ammonium acetate) for tissue sections

  • Commercial autofluorescence reducers like TrueBlack or Vector TrueVIEW

• Optimized Imaging Approaches:

  • Spectral unmixing to separate FITC signal from autofluorescence

  • Time-gated detection to exploit the longer fluorescence lifetime of FITC compared to autofluorescence

  • Confocal microscopy with narrow bandwidth emission filters

  • Consider alternative fluorophores with emission in regions of lower autofluorescence

• Modified Staining Protocols:

  • Extended blocking steps with autofluorescence blockers

  • Higher antibody dilutions with longer incubation times

  • Background subtraction using matched control sections

  • Consider switching to amplification methods like tyramide signal amplification

How should quantitative analysis of FITC-conjugated FABP9 antibody signals be performed?

Quantitative analysis requires rigorous methodological approaches:

• Signal Intensity Quantification:

  • Ensure consistent exposure settings across all samples and controls

  • Perform background subtraction using matched negative controls

  • Use integrated density measurements rather than simple intensity

  • Apply flat-field correction to account for illumination non-uniformity

  • Consider signal-to-noise ratio rather than absolute intensity

• Statistical Approaches:

  • Define regions of interest (ROIs) consistently across samples

  • Include sufficient biological and technical replicates (minimum n=3)

  • Apply appropriate statistical tests based on data distribution

  • Implement randomization and blinding when possible

  • Report variability (standard deviation or standard error) alongside means

• Calibration Methods:

  • Use calibration beads with known fluorophore densities

  • Create standard curves using recombinant FABP9 protein

  • Include internal control proteins with known expression levels

  • Convert fluorescence intensity to relative or absolute protein quantities

• Software Recommendations:

  • ImageJ/FIJI with appropriate plugins for fluorescence quantification

  • CellProfiler for automated high-throughput analysis

  • Specialized single-molecule tracking software for SMT studies

  • Custom MATLAB or Python scripts for complex analyses

What are the best methods for dual or multi-color labeling experiments including FITC-conjugated FABP9 antibodies?

Multi-color immunofluorescence studies require careful planning:

• Fluorophore Selection:

  • Pair FITC (excitation/emission: 495/525 nm) with fluorophores having minimal spectral overlap

  • Good companions include:

    • TRITC or Cy3 (548/562 nm)

    • Cy5 or AlexaFluor 647 (650/668 nm)

    • Pacific Blue (410/455 nm)

  • Avoid strong orange-yellow fluorophores that may overlap with FITC emission

• Controls for Multi-color Experiments:

  • Single-color controls to establish proper compensation/unmixing

  • Fluorescence minus one (FMO) controls to set gating boundaries

  • Secondary antibody-only controls for each fluorophore

  • Sequential staining controls to verify antibody compatibility

• Staining Protocol Optimization:

  • Sequential staining rather than cocktail approaches for challenging targets

  • Careful selection of secondary antibodies raised in different species

  • Blocking between sequential applications to prevent cross-reactivity

  • Order of antibody application (typically from weakest to strongest signal)

• Image Acquisition Strategies:

  • Sequential scanning rather than simultaneous acquisition

  • Linear unmixing to resolve spectral overlap

  • Consistent thresholding approaches across channels

  • Cross-channel bleed-through corrections

How does the F/P ratio of FITC-conjugated antibodies influence experimental outcomes?

The fluorophore-to-protein (F/P) ratio critically impacts experimental results:

• Impact on Binding Properties:

  • Low F/P ratio (<1): May provide insufficient signal for detection

  • Optimal F/P ratio (2-4): Preserves antibody binding capacity while providing sufficient signal

  • High F/P ratio (>6): Can impair antibody binding due to steric hindrance or altered charge, increasing non-specific binding and background

• Effects on Signal Characteristics:

  • Signal intensity generally increases with F/P ratio up to a point

  • Self-quenching occurs at high F/P ratios due to fluorophore proximity

  • Photobleaching rates may increase with higher F/P ratios

  • Signal-to-noise ratio often peaks at intermediate F/P ratios (3-5)

• Application-Specific Considerations:

  • Flow cytometry: Higher F/P ratios (4-6) often acceptable

  • High-resolution microscopy: Lower F/P ratios (1-3) maintain spatial precision

  • Quantitative applications: Consistent F/P ratio between batches is critical

• Determining F/P Ratio:
  Use the equation: Molar F/P = (2.77 × A495) / (A280 – (0.35 × A495))

  Where:

  • A495 is absorbance at 495 nm (FITC absorption maximum)

  • A280 is absorbance at 280 nm (protein absorption maximum)

  • 0.35 is the correction factor for FITC absorption at 280 nm

How can Fab' fragments of FABP9 antibodies improve FITC-conjugated immunofluorescence studies?

Fab' fragments offer several advantages over whole IgG antibodies:

• Improved Spatial Resolution:

  • Smaller size (~50 kDa vs ~150 kDa for whole IgG) reduces the distance between the fluorophore and target

  • Particularly beneficial for super-resolution microscopy techniques

  • Enables more precise protein localization studies

• Reduced Functional Interference:

  • Monovalent binding eliminates crosslinking of target proteins

  • Prevents artificial clustering that can alter protein dynamics

  • Maintains native protein behavior during live-cell imaging

• Enhanced Tissue Penetration:

  • Better penetration into tissues and dense cellular structures

  • Improved access to stereically hindered epitopes

  • More homogeneous staining in thick tissue sections

• Generation Method for Improved Stability:

  • Two-step process to generate stable Fab' fragments:

    1. Digest IgG to F(ab')2 using pepsin

    2. Perform limited reduction with cysteamine to generate Fab' with free sulfhydryl groups in the hinge region

  • This approach maintains the interchain disulfide bond within Fab'

  • Results in more stable conjugates with longer shelf-life than traditional methods

What are the critical parameters for using FITC-conjugated FABP9 antibodies in flow cytometry applications?

Flow cytometry with FITC-conjugated antibodies requires specific optimization:

• Instrument Configuration:

  • Excitation: 488 nm laser line (optimal for FITC)

  • Emission filter: 530/30 nm bandpass filter

  • PMT voltage: Optimize to position negative population within first decade of logarithmic scale

  • Compensation: Adjust for spectral overlap with other fluorophores

• Sample Preparation Considerations:

  • Cell fixation (if needed): 2-4% paraformaldehyde for 10-15 minutes

  • Permeabilization (for intracellular FABP9): 0.1% Triton X-100 or saponin-based buffers

  • Blocking: 5% normal serum from same species as secondary antibody

  • Washing: Multiple washes with 0.1% BSA in PBS to reduce background

• Antibody Titration:

  • Perform serial dilutions to determine saturation point

  • Plot staining index vs. antibody concentration

  • Select concentration at plateau of binding curve

  • Typical dilution range: 1:50 to 1:500 depending on conjugate

• Controls and Analysis Parameters:

  • Unstained cells for autofluorescence assessment

  • Isotype control with matched F/P ratio

  • Single-color controls for compensation setup

  • FMO (Fluorescence Minus One) controls for accurate gating

  • Consider signal-to-noise ratio rather than absolute MFI (Mean Fluorescence Intensity)

How do different fixation methods affect FITC-conjugated FABP9 antibody performance?

Fixation significantly impacts FABP9 detection with FITC-conjugated antibodies:

• Paraformaldehyde Fixation (Cross-linking):

  • Concentration: 2-4% provides optimal balance between structure preservation and epitope accessibility

  • Duration: 10-20 minutes at room temperature

  • Advantages: Good structural preservation, compatible with most epitopes

  • Limitations: May reduce accessibility of some conformational epitopes

  • FITC performance: Generally good signal retention with minimal background

• Methanol/Acetone Fixation (Precipitating):

  • Protocol: Ice-cold methanol or acetone for 5-10 minutes

  • Advantages: Better for certain intracellular epitopes, permeabilizes simultaneously

  • Limitations: Can disrupt membrane structures, may denature some epitopes

  • FITC performance: Can induce higher autofluorescence, especially with acetone

• Hybrid Fixation Methods:

  • Protocol: Brief paraformaldehyde (2%, 10 min) followed by methanol (-20°C, 5 min)

  • Advantages: Combines structural preservation with enhanced epitope accessibility

  • FITC performance: Good compromise for difficult-to-detect epitopes

• Glutaraldehyde Addition:

  • Protocol: Low concentrations (0.05-0.1%) added to paraformaldehyde

  • Advantages: Enhanced structural preservation, especially for cytoskeletal proteins

  • Limitations: Increases autofluorescence significantly

  • FITC performance: Requires careful quenching (e.g., sodium borohydride treatment) to reduce background

• Fresh Frozen Sections:

  • Protocol: Minimal or no fixation, brief acetone post-fixation

  • Advantages: Maximal epitope preservation

  • Limitations: Poor structural preservation

  • FITC performance: Often highest signal intensity but with reduced contextual information