PFAS Antibody, FITC conjugated

What is PFAS (enzyme) and why are antibodies against it important in research?

PFAS (Phosphoribosylformylglycinamidine synthase) is a highly conserved enzyme that catalyzes the fourth step of de novo purine synthesis. It catalyzes the ATP-dependent conversion of formylglycinamide ribonucleotide (FGAR) and glutamine to yield formylglycinamidine ribonucleotide (FGAM) and glutamate . This enzyme plays a critical role in cellular metabolism, particularly in rapidly dividing cells with high nucleotide demands.

Antibodies against PFAS are valuable research tools for:

• Studying purine metabolism disorders

• Investigating cancer cell metabolism

• Examining cellular responses to metabolic stress

• Tracking protein expression across different cell and tissue types

What are the key differences between FITC-conjugated PFAS antibodies and unconjugated variants?

FITC-conjugated PFAS antibodies have the following distinctive characteristics compared to unconjugated versions:

The conjugation process involves attaching FITC to the antibody structure without compromising its binding specificity, allowing direct visualization of PFAS in experimental systems .

What are the recommended applications for PFAS antibody, FITC conjugated?

Based on product information across multiple suppliers, FITC-conjugated PFAS antibodies are recommended for:

It is strongly recommended to optimize the antibody dilution for each specific experimental setup and sample type . Positive control samples reported in the literature include HEK-293T cells, HeLa cells, Jurkat cells, K-562 cells, NIH/3T3 cells, HepG2 cells, and A431 cells .

What are the proper storage and handling conditions for PFAS antibodies with FITC conjugation?

Proper storage and handling are crucial for maintaining antibody activity:

• Storage temperature: Store at -20°C

• Buffer composition: Typically provided in PBS with 0.02-0.03% Proclin 300 and 50% glycerol at pH 7.3-7.4

• Light exposure: Must be protected from light to prevent photobleaching of FITC

• Aliquoting: Recommended for antibodies intended for long-term storage, though some products specify that aliquoting is unnecessary for -20°C storage

• Freeze/thaw cycles: Avoid repeated freeze/thaw cycles

• Stability: Most products are stable for one year after shipment when stored properly

For smaller volume products (20μl sizes), manufacturers often include 0.1% BSA in the storage buffer for additional stability .

What methodological approaches should be used to validate specificity of PFAS antibodies in experimental systems?

A robust validation strategy for PFAS antibodies should include:

• Western Blot Analysis:

  • Confirm single band at expected molecular weight (140-150 kDa)

  • Compare across multiple cell lines known to express PFAS

  • Include negative controls (knockdown/knockout if available)

• Immunohistochemistry Controls:

  • Peptide competition assays - pre-incubating antibody with immunizing peptide should abolish signal

  • Isotype controls - use same host species IgG at matching concentration

  • Cross-reactivity testing across species if using in non-human models

• Flow Cytometry Validation:

  • Compare signal intensity to isotype control

  • Perform blocking experiments with unconjugated primary

  • Use selective cell populations with known expression levels

• Immunofluorescence Specificity:

  • Co-localization with alternative PFAS antibodies targeting different epitopes

  • siRNA knockdown to demonstrate signal reduction

  • Signal pattern should match known subcellular localization

Research indicates that PFAS predominantly localizes within cytoplasmic compartments, with a typical molecular weight in the 140-150 kDa range when detected via Western blot .

How can FITC-conjugated PFAS antibodies be optimized for multi-parameter flow cytometry experiments?

When designing multi-parameter flow cytometry experiments with FITC-conjugated PFAS antibodies:

• Panel Design Considerations:

  • Account for FITC spectral properties (Ex: 499nm, Em: 515nm)

  • Avoid fluorophores with significant spillover into FITC channel (PE, GFP)

  • Consider bright fluorophores for low-expression targets, reserving FITC for higher-expression proteins like PFAS

• Optimization Protocol:

  • Titrate antibody with 3-5 concentrations across recommended range (1:50-1:200)

  • Calculate staining index for each concentration: (MFI positive - MFI negative)/2 × SD of negative

  • Select concentration with highest staining index and acceptable background

• Compensation Strategy:

  • Prepare single-stained controls for each fluorophore

  • Include FMO (Fluorescence Minus One) control omitting FITC-PFAS antibody

  • Use automated compensation with manual adjustment if necessary

• Sample Preparation Refinements:

  • Optimize fixation protocol (PFA concentration and duration)

  • Test different permeabilization reagents (Triton X-100, saponin, methanol)

  • Block with species-appropriate serum (5-10%) to reduce non-specific binding

Several manufacturers report successful application of PFAS antibodies in flow cytometry, particularly with human cell lines including HEK-293T, HeLa, and Jurkat cells .

What are the methodological considerations for using PFAS antibodies in quantitative immunofluorescence studies?

For quantitative analysis of PFAS expression using immunofluorescence:

• Image Acquisition Parameters:

  • Establish consistent exposure settings based on brightest sample

  • Capture multiple random fields to account for heterogeneity

  • Include calibration standards for absolute quantification

  • Set z-stack parameters to capture full cellular volume

• Signal Quantification Approach:

  • Calculate mean fluorescence intensity (MFI) within defined cellular regions

  • Measure nuclear/cytoplasmic ratio to assess relative distribution

  • Apply background subtraction using cell-free regions

  • Consider signal density (intensity/area) for morphologically diverse samples

• Validation Controls:

  • Serially diluted recombinant PFAS protein as standard curve

  • Parallel analysis with alternative detection methods (e.g., Western blot)

  • Include samples with known differential expression

• Technical Optimization:

  • Test multiple antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Compare mounting media for optimal signal preservation

  • Validate linearity of signal across protein concentration range

Published research demonstrates successful localization of PFAS in cell lines including A431 and HepG2 cells using immunofluorescence techniques .

How do different sample preparation methods affect FITC-conjugated PFAS antibody performance in immunocytochemistry?

Sample preparation significantly impacts FITC-conjugated PFAS antibody staining outcomes:

Permeabilization Optimization:

• Triton X-100 (0.1-0.5%): Effective for nuclear proteins, may extract some cytoplasmic components

• Saponin (0.1-0.3%): Gentler permeabilization, better preservation of membrane structures

• Digitonin (10-50 μg/ml): Selective permeabilization of plasma membrane, preserves nuclear envelope

Blocking Strategy:
For optimal signal-to-noise ratio, implement this protocol:

• Fix cells using 4% PFA for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 10 minutes

• Block with 5% normal serum (from secondary antibody host species) for 30-60 minutes

• Apply FITC-conjugated PFAS antibody at 1:200 dilution in blocking buffer

• Incubate overnight at 4°C protected from light

• Wash extensively (3-5 times) with PBS containing 0.05% Tween-20

This methodology has been successfully applied with A431 cells in published reports .

What are the technical challenges and solutions when using PFAS antibodies for detecting the enzyme versus developing immunoassays for environmental PFAS contaminants?

This question addresses an important distinction between two different applications of "PFAS" terminology:

For environmental PFAS detection, researchers have developed approaches using peroxisome proliferator-activated receptor alpha (PPARα) as a bioreceptor, since PPARα binds to many PFAS compounds. This allows for simultaneous detection of multiple environmental PFAS contaminants . In contrast, enzyme PFAS antibodies typically target specific epitopes within the large phosphoribosylformylglycinamidine synthase protein .

How can PFAS antibodies contribute to understanding immunotoxicity mechanisms of environmental PFAS compounds?

Research on the immunotoxicity of environmental PFAS compounds can be enhanced using antibody-based approaches:

• Mechanism Investigation Approaches:

  • Using antibodies to detect changes in immune cell protein expression following PFAS exposure

  • Quantifying B-cell subset alterations using multi-parameter flow cytometry with appropriate markers

  • Tracking plasmablast development and antibody-secreting cell formation with cell-type specific markers

• Experimental Design Framework:

  • Control vs. PFAS-exposed comparison (animal models show effects at 7.5 mg/kg dosing)

  • Dose-response relationships (critical for regulatory assessments)

  • Time-course experiments to identify acute vs. chronic effects

  • Sex-stratified analysis (documented sex-specific differences in responses)

• Key Findings from Published Literature:

  • PFOA exposure (7.5 mg/kg) decreased numbers of plasmablasts, follicular B cells, and naïve B cells in female mice

  • Male mice showed different patterns with increases in follicular B cells but decreases in other subsets

  • Prenatal PFAS exposure associated with reduced antibody responses to vaccination

  • Log-unit increase in perfluorononanoate associated with 2.09 times higher odds of low measles antibody titers

  • Log-unit increase in perfluorooctanoate associated with 2.46 times higher odds of low mumps antibody titers

• Methodological Approaches:

  • Flow cytometry panels for immune cell subset characterization

  • ELISA-based quantification of vaccine-specific antibody responses

  • Metabolic profiling of immune cells following PFAS exposure

  • Ex vivo B-cell activation assays to assess functional responses

Research has demonstrated that PFAS exposure can alter specific B-cell populations and affect antibody responses to vaccines, with potential implications for public health .

What are the best practices for multiplexed imaging using FITC-conjugated PFAS antibodies alongside other fluorophores?

Successful multiplexed imaging requires careful fluorophore selection and protocol optimization:

• Fluorophore Selection Strategy:

  • Pair FITC (Em max: 515nm) with spectrally distant fluorophores such as:

    • DAPI (Em max: ~460nm) for nuclear counterstaining

    • Cy3/TRITC (Em max: ~570nm) for secondary target

    • Cy5/APC (Em max: ~670nm) for tertiary target

  • Consider brightness hierarchy: assign brightest fluorophores to lowest-expressing targets

• Sequential Staining Protocol for Co-localization Studies:
  a) Apply unconjugated primary antibody for first target
  b) Add appropriate secondary antibody (non-FITC)
  c) Block with excess unconjugated host IgG
  d) Apply FITC-conjugated PFAS antibody
  e) Counterstain nuclei with DAPI

• Image Acquisition Parameters:

  • Capture single-fluorophore controls to assess bleed-through

  • Image sequentially rather than simultaneously when possible

  • Apply appropriate exposure settings to prevent saturation

  • Include no-primary controls for each fluorophore channel

• Co-localization Analysis Approaches:

  • Calculate Pearson's or Manders' coefficients for quantitative assessment

  • Apply appropriate thresholding to exclude background

  • Consider object-based co-localization for discrete structures

Manufacturers report successful application in immunofluorescence with A431 cells and HepG2 cells at dilutions ranging from 1:50-1:800 .

How can researchers integrate PFAS antibody-based detection with metabolic studies of purine biosynthesis?

Integrating PFAS detection with metabolic studies requires a multi-disciplinary approach:

• Experimental Design Framework:

  • Parallel analysis of PFAS protein levels and metabolic intermediates

  • Time-course studies capturing dynamic changes in enzyme expression

  • Perturbation experiments using purine synthesis inhibitors or nutrient limitation

  • Correlation of PFAS localization with metabolic activity markers

• Integrated Method Protocol:
  a) Divide cell/tissue samples for parallel processing:

  • Portion 1: Protein extraction for PFAS quantification (Western blot/ELISA)

  • Portion 2: Metabolite extraction for purine intermediate analysis (LC-MS/MS)

  • Portion 3: Fixation for spatial analysis (immunofluorescence)
    b) Normalize data to cell number or protein content
    c) Perform correlation analysis between PFAS levels and metabolite concentrations

• Functional Validation Approaches:

  • PFAS knockdown/knockout to confirm metabolic consequences

  • Rescue experiments with wildtype vs. mutant PFAS

  • Metabolic flux analysis using isotope-labeled precursors

  • Live-cell imaging of PFAS dynamics during metabolic challenges

PFAS catalyzes the ATP-dependent conversion of formylglycinamide ribonucleotide (FGAR) and glutamine to yield formylglycinamidine ribonucleotide (FGAM) and glutamate . This represents a critical step in de novo purine synthesis that can be monitored alongside enzyme expression levels.