PAFAH2 Antibody, FITC conjugated

What is PAFAH2 and why is it important in research?

PAFAH2 (Platelet-Activating Factor Acetylhydrolase 2, 40kDa) is a cytoplasmic enzyme that catalyzes the degradation of platelet-activating factor by hydrolyzing the sn-2 acetyl ester bond. This protein plays significant roles in inflammatory processes, lipid metabolism, and cellular signaling pathways. The study of PAFAH2 is critical for understanding various physiological and pathological conditions, including inflammatory disorders, cardiovascular diseases, and certain neurological conditions. Researchers commonly use antibodies against PAFAH2 to investigate its expression, localization, and functional relationships in various experimental systems .

What are the key specifications of commercially available FITC-conjugated PAFAH2 antibodies?

Currently available FITC-conjugated PAFAH2 antibodies typically feature the following specifications:

• Target epitope: Most commonly targeting amino acids 1-206 of the human PAFAH2 protein

• Host species: Predominantly rabbit-derived

• Clonality: Primarily polyclonal antibodies

• Reactivity: Human-specific, though some products may cross-react with mouse or rat

• Purity: Generally >95% pure, purified via Protein G or immunogen affinity methods

• Immunogen: Recombinant human PAFAH2 protein fragments (commonly amino acids 1-206)

• Isotype: IgG

• Applications: Compatible with immunofluorescence (IF), immunohistochemistry (IHC), and ELISA methodologies

What is the significance of FITC conjugation for PAFAH2 antibodies?

FITC (Fluorescein Isothiocyanate) conjugation attaches a bright green fluorescent dye directly to the antibody molecule, enabling direct visualization of target antigens without requiring secondary detection reagents. This conjugation offers several experimental advantages:

• Simplified protocols by eliminating secondary antibody incubation steps

• Reduced background signal in multi-color immunofluorescence experiments

• Direct quantification of protein expression using flow cytometry

• Enhanced sensitivity in detecting low-abundance proteins

• Compatibility with live-cell imaging applications

The excitation maximum for FITC is approximately 495 nm, and emission maximum is around 520 nm, making it compatible with standard FITC/GFP filter sets on most fluorescence microscopes and flow cytometers .

What are the optimal dilution factors for FITC-conjugated PAFAH2 antibodies in different applications?

Based on manufacturer specifications and research protocols, the recommended dilution ranges for FITC-conjugated PAFAH2 antibodies are:

These dilution factors should serve as starting points, and researchers should perform titration experiments to determine optimal conditions for their specific experimental systems .

How does the binding specificity to amino acids 1-206 of PAFAH2 influence experimental applications?

The binding specificity to amino acids 1-206 of PAFAH2 has several important implications for research applications:

• This region contains essential functional domains of the protein, making the antibody useful for studying protein activity and interactions

• The N-terminal region (1-206) is more accessible in native protein conformations, enhancing detection in non-denaturing conditions

• This specificity may limit detection of certain splice variants or processed forms of PAFAH2 that lack this region

• Cross-reactivity with other proteins is minimized due to the specificity of this epitope region

• The 1-206 region is highly conserved across species, potentially enabling cross-species applications despite manufacturer specifications focusing on human reactivity

Researchers should consider these factors when designing experiments, particularly when studying protein-protein interactions or conformational changes of PAFAH2.

What are the optimal conditions for conjugating FITC to PAFAH2 antibodies?

For researchers performing custom FITC conjugation to PAFAH2 antibodies, the following conditions have been empirically determined to yield optimal results:

• Reaction time: 30-60 minutes

• Temperature: Room temperature (20-25°C)

• pH: 9.5 (optimal for the conjugation reaction)

• Initial protein concentration: 25 mg/ml

• Buffer system: Carbonate-bicarbonate buffer

These conditions maximize the fluorescein/protein (F/P) ratio while maintaining antibody activity. Following conjugation, separation of optimally labeled antibodies from under- and over-labeled proteins is achievable through gradient DEAE Sephadex chromatography .

What factors affect the stability and performance of FITC-conjugated PAFAH2 antibodies?

Several critical factors influence the stability and performance of FITC-conjugated PAFAH2 antibodies:

• Light exposure: FITC is susceptible to photobleaching; store and handle in dark conditions

• pH sensitivity: FITC fluorescence decreases significantly below pH 7.0

• Storage temperature: Optimal stability at 2-8°C; avoid freeze-thaw cycles

• Protein concentration: Higher concentrations (>1 mg/ml) generally provide better stability

• Buffer composition: Presence of protein stabilizers (e.g., BSA) and preservatives improves long-term stability

• Degree of labeling: Over-labeling can cause self-quenching and reduced antibody affinity

To maximize performance, store FITC-conjugated PAFAH2 antibodies in amber vials at 2-8°C protected from light, and avoid repeated freeze-thaw cycles .

How can researchers validate the specificity of FITC-conjugated PAFAH2 antibodies?

Rigorous validation ensures experimental reliability when working with FITC-conjugated PAFAH2 antibodies. Recommended validation approaches include:

• Positive controls: Test on cell lines or tissues known to express PAFAH2 (e.g., HepG2, placental tissue)

• Negative controls: Test on cell lines with PAFAH2 knockdown or tissues known not to express PAFAH2

• Blocking peptide controls: Pre-incubate antibody with immunizing peptide to confirm specificity

• Western blot confirmation: Perform parallel Western blot (with unconjugated version of the same antibody) to confirm target molecular weight (~40 kDa)

• Multi-antibody validation: Compare results with different PAFAH2 antibodies targeting distinct epitopes

• Orthogonal methods: Compare expression data with mRNA expression (RT-PCR or RNA-seq)

This multi-faceted validation approach helps establish confidence in antibody specificity before proceeding with comprehensive experimental studies .

What are common issues with FITC-conjugated PAFAH2 antibodies and how can they be resolved?

Implementing these troubleshooting strategies should address most common issues encountered with FITC-conjugated PAFAH2 antibodies .

How can researchers optimize dual/multi-label immunofluorescence experiments involving FITC-conjugated PAFAH2 antibodies?

When incorporating FITC-conjugated PAFAH2 antibodies into multi-color immunofluorescence experiments, consider these optimization strategies:

• Fluorophore selection: Choose companion fluorophores with minimal spectral overlap with FITC (good options include TRITC/Texas Red and far-red dyes like Cy5)

• Sequential staining: Apply antibodies sequentially rather than simultaneously to minimize cross-reactivity

• Blocking optimization: Use species-specific blocking reagents matched to each primary antibody

• Antibody order: Apply the FITC-conjugated PAFAH2 antibody last in the sequence to minimize photobleaching

• Controls: Include single-color controls to establish bleed-through parameters for compensation

• Image acquisition: Capture FITC channel first, as it is more susceptible to photobleaching than other fluorophores

• Microscopy settings: Optimize exposure settings individually for each channel to balance signal intensity

These approaches help maximize signal quality while minimizing artifacts in multi-color experiments involving FITC-conjugated PAFAH2 antibodies .

How should researchers quantify and interpret fluorescence signals from FITC-conjugated PAFAH2 antibodies?

Accurate quantification of FITC-conjugated PAFAH2 antibody signals requires systematic analytical approaches:

• Image acquisition standardization:

  • Use consistent exposure settings across all experimental conditions

  • Ensure signals are within the linear range of detection (not saturated)

  • Capture multiple representative fields per sample

• Background subtraction methods:

  • Subtract autofluorescence using unstained controls

  • Apply local background subtraction algorithms

• Quantification approaches:

  • For cellular localization: Calculate nuclear/cytoplasmic ratio of fluorescence intensity

  • For expression levels: Measure integrated density or mean fluorescence intensity

  • For co-localization: Calculate Pearson's or Mander's coefficient with co-stained markers

• Data normalization:

  • Normalize to housekeeping protein expression

  • Compare relative rather than absolute fluorescence values between experiments

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Consider biological replicates rather than technical replicates for statistical power

These methodological approaches ensure reliable quantification and meaningful interpretation of FITC-conjugated PAFAH2 antibody signals .

What controls are essential when using FITC-conjugated PAFAH2 antibodies?

A comprehensive control strategy ensures reliable results when working with FITC-conjugated PAFAH2 antibodies:

• Primary controls:

  • Isotype control: FITC-conjugated rabbit IgG at the same concentration

  • Absorption control: PAFAH2 antibody pre-incubated with excess antigen

  • Genetic controls: PAFAH2 knockout or knockdown samples

• Technical controls:

  • Autofluorescence control: Unstained sample to establish baseline fluorescence

  • Secondary-only control: When using unconjugated primary antibodies

  • Fixation control: Samples processed identically but without antibody

• Specificity controls:

  • Cross-validation with alternative PAFAH2 antibodies

  • Correlation with PAFAH2 mRNA expression

  • Western blot confirmation of specificity

• Quantification controls:

  • Fluorescence standards for inter-experimental calibration

  • Internal reference cells/tissues with known PAFAH2 expression levels

Implementing this control strategy provides the necessary framework for robust experimental design and data interpretation when working with FITC-conjugated PAFAH2 antibodies .