SRPX Antibody, FITC conjugated

What is SRPX Antibody and what cellular functions does it target?

SRPX antibody targets the Sushi repeat-containing protein SRPX (also known as ETX1), which is involved in several critical cellular processes. According to current research, SRPX protein may play significant roles in phagocytosis during disk shedding, cell adhesion to cells other than the pigment epithelium, and signal transduction pathways . The protein has a UniProt ID of P78539 and contains specific structural domains that enable its diverse cellular functions.

When selecting an SRPX antibody for research, it's important to understand that different antibodies may target different epitopes of the protein, potentially affecting experimental outcomes. Antibodies raised against the recombinant human SRPX protein, particularly regions spanning amino acids 31-237, have demonstrated high specificity in laboratory applications . For comprehensive characterization of SRPX functions, researchers should consider employing multiple antibodies targeting different protein domains.

What is FITC conjugation and how does it enhance antibody functionality?

FITC (Fluorescein isothiocyanate) conjugation involves the covalent attachment of FITC molecules to antibodies through a reaction with free amino groups of proteins, forming stable conjugates . This chemical process creates antibodies that can be directly visualized using fluorescence detection systems without requiring secondary antibodies.

FITC is among the most widely used fluorescent labeling reagents due to its high quantum efficiency and the stability of the resulting conjugates . The fluorophore has an absorption maximum at 495 nm and emission maximum at 525 nm, producing a yellow-green fluorescence when excited with ultraviolet or blue light . This spectral profile makes FITC-conjugated antibodies ideally suited for multicolor flow cytometry and fluorescence microscopy applications.

The conjugation of FITC to antibodies enables direct visualization of target antigens in various experimental systems including immunohistochemistry, immunofluorescence, and flow cytometry. The primary advantage is the elimination of additional detection steps that would be required with unconjugated primary antibodies, thereby simplifying protocols and potentially reducing background interference.

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

For maximum stability and retention of activity, SRPX Antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt . The liquid form of the antibody is typically preserved in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . This formulation helps maintain antibody integrity during freeze-thaw cycles.

It is critically important to avoid repeated freeze-thaw cycles, as these can significantly degrade antibody performance. Best practice involves aliquoting the antibody into multiple vials upon receipt to minimize the number of freeze-thaw events for any single sample . When planning experiments, researchers should remove only the amount needed for immediate use and keep the remainder frozen.

For short-term storage (up to one week), the antibody can be kept at 2-8°C, protected from light to prevent photobleaching of the FITC fluorophore. Extended exposure to light should be avoided at all stages of handling, as this can diminish the fluorescence intensity and compromise experimental results.

How does the FITC labeling index affect antibody binding affinity and specificity?

The FITC labeling index, defined as the molar ratio of FITC molecules per antibody molecule (F/P ratio), critically influences both the binding affinity and specificity of the conjugated antibody. Research has demonstrated that this relationship is inversely proportional—higher labeling indices correlate with decreased binding affinity for target antigens . This phenomenon occurs because excessive FITC conjugation can modify amino acid residues within or adjacent to the antigen-binding sites.

In immunohistochemical applications, antibodies with higher labeling indices generally exhibit increased sensitivity but are also more prone to generating non-specific staining . This creates a technical paradox where researchers must balance detection sensitivity against signal specificity. The optimal F/P ratio typically falls within the range of 3-6 FITC molecules per antibody, which preserves sufficient binding affinity while providing adequate fluorescence intensity.

Experimental evidence indicates that when the F/P ratio exceeds 6:1, several adverse effects may occur, including:

• Increased non-specific binding leading to higher background fluorescence

• Decreased quantum yield due to self-quenching effects between closely positioned FITC molecules

• Potential antibody aggregation or precipitation

• Altered binding kinetics that may compromise quantitative analyses

For critical applications requiring precise quantification, researchers should verify the F/P ratio of their FITC-conjugated antibodies and validate performance against appropriate controls before proceeding with full-scale experiments.

What are the optimal conditions for FITC conjugation to maximize antibody activity?

The conjugation of FITC to antibodies requires careful optimization of multiple parameters to preserve antibody functionality while achieving sufficient fluorescence labeling. Based on comprehensive experimental analyses, the following conditions have been identified as optimal for FITC conjugation:

• pH conditions: A reaction pH of 9.5 provides the optimal environment for FITC conjugation, as it enhances the reactivity of amino groups on the antibody . Most conjugation protocols utilize a 0.1M carbonate-bicarbonate buffer system to maintain this alkaline pH.

• Reaction temperature: Room temperature (20-25°C) has been demonstrated to yield efficient conjugation while minimizing protein denaturation . Higher temperatures can accelerate the reaction but risk compromising antibody structure.

• Protein concentration: An initial protein concentration of approximately 25 mg/ml has been shown to achieve maximal labeling efficiency . Lower concentrations may result in inconsistent labeling.

• Reaction time: Optimal labeling can be achieved within 30-60 minutes under the conditions described above . Extending the reaction time beyond this window rarely improves labeling efficiency and may increase the risk of over-labeling.

• FITC:antibody molar ratio: To determine the optimal ratio for a specific antibody, it is advisable to perform small-scale conjugations at different molar ratios (typically 10:1, 20:1, and 30:1) before proceeding to larger preparations . The ratio that yields the most favorable F/P ratio (typically 3-6) while maintaining antibody activity should be selected.

Following conjugation, purification via gradient DEAE Sephadex chromatography can effectively separate optimally labeled antibodies from under- and over-labeled proteins, ensuring a more homogeneous product with consistent performance characteristics .

How can researchers troubleshoot non-specific binding when using SRPX Antibody, FITC conjugated?

Non-specific binding represents one of the most common challenges when working with FITC-conjugated antibodies, including SRPX antibody. This phenomenon manifests as background fluorescence that reduces signal-to-noise ratio and complicates data interpretation. Several methodological approaches can effectively mitigate this issue:

• Optimize blocking conditions: Increase the concentration of blocking proteins (BSA, normal serum, or commercial blocking reagents) to 5-10% and extend the blocking time to 1-2 hours at room temperature. This helps saturate potential non-specific binding sites before antibody application.

• Dilution series optimization: Perform a systematic titration of the FITC-conjugated SRPX antibody, testing dilutions from 1:20 to 1:200 for applications such as immunofluorescence and flow cytometry . The optimal dilution provides sufficient specific signal while minimizing background.

• Include appropriate controls: Always incorporate an isotype control antibody with matching FITC labeling index to distinguish between specific and non-specific binding . For SRPX antibody raised in rabbit, a FITC-conjugated rabbit IgG isotype control should be employed at the same concentration.

• Pre-adsorption with target tissues: When experiencing persistent cross-reactivity, pre-adsorbing the antibody with cells or tissues lacking the target antigen can reduce non-specific interactions.

• Buffer optimization: The addition of 0.1-0.5% non-ionic detergents (such as Tween-20) to washing buffers can significantly reduce hydrophobic interactions contributing to background. Additionally, including 0.1-0.3M NaCl in antibody diluent can disrupt low-affinity electrostatic interactions.

• Sample processing considerations: For fixed samples, autofluorescence can be reduced by treating with 0.1% sodium borohydride or commercial autofluorescence reducers prior to antibody incubation.

Implementing these approaches systematically can substantially improve signal specificity in experiments utilizing FITC-conjugated SRPX antibody, enabling more accurate interpretation of results.

What are the validated applications for SRPX Antibody, FITC conjugated?

When adapting the antibody for applications beyond ELISA, researchers should perform preliminary validation studies incorporating appropriate positive and negative controls to confirm specificity and determine optimal working concentrations. The antibody's reactivity to human samples has been established , but cross-reactivity with samples from other species should be empirically determined before proceeding with full experiments.

How should researchers optimize SRPX Antibody, FITC conjugated for flow cytometry?

Optimizing SRPX Antibody, FITC conjugated for flow cytometry requires systematic protocol development to ensure reliable detection of target antigens while minimizing background interference. A methodical approach includes:

• Antibody titration: Create a dilution series (typically 1:20, 1:50, 1:100) to identify the concentration that produces maximum signal separation between positive and negative populations . The optimal concentration is often defined as the highest dilution that maintains a robust stain index.

• Sample preparation optimization: For intracellular targets, evaluate different fixation and permeabilization methods, as these can significantly impact epitope accessibility. For SRPX detection, start with standard formaldehyde fixation (2-4%) followed by permeabilization with 0.1% Triton X-100 or commercially available permeabilization buffers.

• Compensation setup: As FITC's emission spectrum (emission max: 525 nm) may overlap with other fluorophores, proper compensation is essential for multicolor panels . Use single-stained controls for each fluorophore to establish compensation matrices.

• Dead cell discrimination: Include a viability dye compatible with FITC (avoiding spectral overlap) to exclude dead cells, which can bind antibodies non-specifically.

• Gating strategy development: Design a sequential gating strategy that:

  • Excludes debris based on forward/side scatter

  • Removes doublets using height vs. area measurements

  • Eliminates dead cells

  • Applies appropriate positive/negative gates based on FMO (Fluorescence Minus One) controls

• Controls implementation:

  • Unstained samples to establish autofluorescence baseline

  • FITC-conjugated isotype control at the same concentration as the SRPX antibody

  • FMO controls for complex panels

  • Positive control samples with known SRPX expression

• Instrument settings optimization: Adjust PMT voltages to position the negative population appropriately on scale while ensuring the positive population remains within the detector's linear range.

Following this systematic approach will facilitate reliable detection of SRPX in flow cytometry applications while minimizing artifacts and background interference.

What controls should be included when using SRPX Antibody, FITC conjugated in immunofluorescence microscopy?

Implementing a comprehensive control strategy is essential for generating reliable and interpretable data when using SRPX Antibody, FITC conjugated in immunofluorescence microscopy. The following controls should be included in experimental design:

• Primary antibody controls:

  • Isotype control: A FITC-conjugated rabbit IgG isotype control at the same concentration as the SRPX antibody to assess non-specific binding

  • Concentration gradient: Series of primary antibody dilutions (e.g., 1:50, 1:100, 1:200) to determine optimal signal-to-noise ratio

• Sample preparation controls:

  • Autofluorescence control: Sample processed identically but without any antibody to assess intrinsic fluorescence

  • Secondary antibody-only control: Only relevant when using additional detection systems

• Biological controls:

  • Positive control: Cell line or tissue with confirmed SRPX expression

  • Negative control: Cell line or tissue with confirmed absence of SRPX expression

  • Knockdown/knockout validation: When available, samples with SRPX gene silencing provide stringent specificity controls

• Technical controls:

  • Absorption control: SRPX antibody pre-incubated with excess target antigen to confirm binding specificity

  • Cross-reactivity assessment: Testing the antibody on tissues from different species if cross-species reactivity is claimed

• Imaging controls:

  • Exposure settings: Standardized across all samples and controls

  • Channel bleed-through check: Especially important in multi-color experiments

  • Z-stack acquisition: For three-dimensional localization studies

• Data analysis controls:

  • Quantification standards: Calibrated fluorescence standards for quantitative analyses

  • Blinded analysis: Samples coded to prevent observer bias during analysis

The implementation of this control framework enables confident differentiation between specific SRPX staining and various sources of background or artifact, substantially improving data quality and interpretability in immunofluorescence applications.

How does the fluorescein/protein (F/P) ratio impact experimental outcomes?

The fluorescein/protein (F/P) ratio, which quantifies the number of FITC molecules conjugated to each antibody molecule, substantially influences experimental outcomes across multiple dimensions. Understanding these effects enables researchers to select appropriate antibody preparations for specific applications:

When using commercially available FITC-conjugated antibodies, researchers should consult product specifications for F/P ratio information. For custom conjugations, small-scale pilot studies with different FITC:antibody ratios are recommended to identify optimal preparation conditions for specific experimental requirements .

What methods are available for determining the F/P ratio of FITC-conjugated antibodies?

Accurate determination of the fluorescein/protein (F/P) ratio is essential for standardizing experiments and interpreting results obtained with FITC-conjugated antibodies. Several methodological approaches with varying complexity and precision are available to researchers:

• Spectrophotometric method: This widely used approach leverages the distinct absorption characteristics of FITC and proteins:

  • Measure absorbance at 280 nm (A₂₈₀) for protein content and 495 nm (A₄₉₅) for FITC content

  • Calculate F/P ratio using the formula:
    F/P ratio = (A₄₉₅ × dilution factor) / [(A₂₈₀ - (0.35 × A₄₉₅)) × ε₂₈₀]

  • Where ε₂₈₀ is the molar extinction coefficient of the antibody at 280 nm

  • The factor 0.35 accounts for FITC contribution to absorbance at 280 nm

• Gradient DEAE Sephadex chromatography: This method not only determines F/P ratio but also enables separation of optimally labeled antibodies from under- and over-labeled proteins . The technique utilizes differential binding of conjugates based on charge modifications introduced by FITC labeling.

• Mass spectrometry: For high-precision determination, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry can directly measure the mass difference between unlabeled and FITC-labeled antibodies, providing accurate F/P ratios.

• Fluorescence correlation spectroscopy (FCS): This advanced biophysical technique can determine the average number of fluorescent molecules per diffusing particle, offering a solution-based approach to F/P ratio determination.

• Commercial F/P determination kits: Several manufacturers offer standardized kits that include calibrated FITC standards and detailed protocols for F/P ratio determination.

The selection of an appropriate method depends on available instrumentation, required precision, and sample quantity. For routine research applications, the spectrophotometric method offers a reasonable balance of accessibility and accuracy. For critical applications requiring precise quantification or preparation of reference standards, orthogonal methods such as mass spectrometry or FCS provide enhanced precision at the cost of increased technical complexity.

What are the advantages and limitations of FITC-conjugated primary antibodies versus two-step detection systems?

The choice between directly FITC-conjugated primary antibodies (like SRPX Antibody, FITC conjugated) and two-step detection systems (unconjugated primary antibody followed by FITC-conjugated secondary antibody) represents a significant methodological decision that impacts experimental outcomes. A comparative analysis reveals distinct advantages and limitations of each approach:

FITC-Conjugated Primary Antibodies:

Advantages:

Limitations:

• Reduced signal amplification compared to secondary detection systems

• Potential reduction in primary antibody binding affinity due to FITC conjugation

• Limited flexibility in signal optimization post-conjugation

• Higher cost per experiment due to larger quantities of specialized conjugated antibody

• Fluorescence photobleaching directly affects primary antibody detection capacity

Two-Step Detection Systems:

Advantages:

• Signal amplification through multiple secondary antibody binding to each primary antibody

• Preservation of primary antibody binding affinity

• Flexibility to change detection system (fluorophore) without changing primary antibody

• More cost-effective for large experimental series

• Ability to implement tyramide signal amplification for ultra-sensitive detection

Limitations:

• More complex protocols with additional incubation and washing steps

• Increased risk of non-specific binding and background

• Potential cross-reactivity issues in multiple labeling experiments

• Extended protocol duration

• Greater susceptibility to washing efficiency variations

For optimal experimental design, researchers should consider several factors including target abundance, sample type, required sensitivity, and experimental constraints. FITC-conjugated primary antibodies like SRPX Antibody, FITC conjugated are particularly advantageous for:

• Targets with high expression levels

• Applications requiring minimal protocol duration

• Multicolor immunofluorescence with antibodies from limited species options

• Samples with high endogenous immunoglobulin content

Conversely, two-step detection systems are preferable for low-abundance targets, where signal amplification can make the difference between detection and false negatives.

How can SRPX Antibody, FITC conjugated be utilized in multiplex immunofluorescence assays?

Multiplex immunofluorescence assays enable simultaneous visualization of multiple targets within a single sample, providing valuable insights into protein co-localization and spatial relationships. Integrating SRPX Antibody, FITC conjugated into multiplex panels requires careful consideration of spectral properties, panel design, and protocol optimization:

• Spectral considerations: FITC has excitation/emission maxima at approximately 495/519 nm , producing yellow-green fluorescence. When designing multiplex panels:

  • Pair FITC with fluorophores having minimal spectral overlap such as DAPI (blue), Cy3 (orange), and Cy5 (far-red)

  • Avoid combining with PE (phycoerythrin) due to significant spectral overlap

  • Consider the specific filter sets available on imaging platforms

• Panel design strategies:

  • Begin with the lowest abundance target in the brightest channel

  • Consider the biological relationship between targets (co-localization expectations)

  • Balance nuclear, cytoplasmic, and membrane markers across different channels

  • Include one marker per cellular compartment for initial spatial registration

• Sequential immunostaining approach:

  • For complex panels (>3 markers), sequential staining often provides superior results

  • Begin with heat-induced epitope retrieval optimized for all targets

  • Apply multiple primary-secondary pairs sequentially with microwave treatment between rounds

  • For each round:

    • Apply primary antibody

    • Apply compatible fluorophore-conjugated secondary

    • Microwave to remove previous antibodies while preserving fluorophores

    • Proceed to next marker

• Direct conjugate optimization:

  • When using multiple directly-conjugated antibodies including SRPX Antibody, FITC conjugated:

    • Balance concentrations based on target abundance and fluorophore brightness

    • Apply simultaneously unless specific ordering is required

    • Implement longer incubation times (overnight at 4°C) to maximize sensitivity

• Validation strategies:

  • Single-color controls to assess bleed-through

  • FMO (Fluorescence Minus One) controls to set proper compensation

  • Biological controls demonstrating expected co-localization patterns

  • Antibody absorption controls to confirm specificity in multiplex context

• Image acquisition considerations:

  • Sequential channel acquisition to minimize bleed-through

  • Consistent exposure settings between samples

  • Z-stack acquisition for co-localization analysis

  • Appropriate background subtraction methods

Successful integration of SRPX Antibody, FITC conjugated into multiplex panels requires systematic optimization but provides uniquely valuable data on spatial relationships between SRPX and other proteins of interest in cellular contexts.