PITPNA Antibody, FITC conjugated

What is PITPNA and what are its primary cellular functions?

PITPNA catalyzes the transfer of phosphatidylinositol (PtdIns) and phosphatidylcholine between membrane compartments, thereby regulating lipid metabolism, membrane trafficking, and signaling in eukaryotic cells . It serves as an essential component of phospholipase C (PLC) signaling, particularly in neurons where it facilitates neurite outgrowth . PITPNA has also been shown to control extension of laminin-dependent axonal processes by regulating phosphatidylinositol 3-kinase (PI3K)-dependent signaling during neurite remodeling . Additionally, PITPNA modulates PI3K/Akt signaling, which is crucial for cell growth, metabolism, and apoptosis .

What distinguishes FITC-conjugated PITPNA antibody from other conjugates?

FITC (fluorescein isothiocyanate) conjugation provides direct fluorescent detection capabilities without requiring secondary antibodies, simplifying immunofluorescence protocols. Unlike biotin-tagged antibodies, FITC-conjugated antibodies avoid potential background issues from endogenous biotin in tissues . The PITPNA antibody with FITC conjugation is typically a rabbit-hosted polyclonal antibody reactive to human samples, purified using antigen affinity methods . This conjugation allows for detection of PITPNA in subcellular compartments with green fluorescence under appropriate excitation wavelengths.

What are the optimal storage and handling conditions for PITPNA Antibody, FITC conjugated?

Store the antibody at -20°C or -80°C to maintain stability and activity . The antibody is supplied in liquid form in a buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . Avoid repeated freeze-thaw cycles as they can damage the antibody and reduce its effectiveness . For short-term storage (1-2 weeks), aliquots may be kept at 4°C. Working dilutions should be prepared fresh before use to ensure optimal performance in experimental applications.

What are the validated research applications for PITPNA Antibody, FITC conjugated?

The antibody has been validated for applications in neuroscience and signal transduction research . Specifically, it can be used to:

• Detect native and recombinant PITPNA in human samples

• Visualize PITPNA distribution in subcellular compartments using immunofluorescence

• Examine PITPNA's role in PI3K/Akt signaling pathways in various cell types

• Investigate PITPNA functions in neuronal development and degeneration models

• Study PITPNA involvement in metabolic disorders like Type 2 diabetes

What protocol is recommended for immunofluorescence with PITPNA Antibody, FITC conjugated?

Recommended Immunofluorescence Protocol:

• Fixation and Permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Wash 3× with PBS

  • Permeabilize with 0.5% Triton X-100 in PBS for 10 minutes

• Blocking:

  • Block with 5% normal serum in PBS for 1 hour at room temperature

• Primary Antibody Incubation:

  • Dilute FITC-conjugated PITPNA antibody in blocking buffer (recommended dilution: 1:50 to 1:200)

  • Incubate overnight at 4°C in a humidified chamber

  • Protect from light during and after this step

• Counterstaining:

  • Wash 3× with PBS

  • Counterstain nuclei with DAPI (1:1000) for 10 minutes

• Mounting and Imaging:

  • Mount with anti-fade mounting medium

  • Image using appropriate filters for FITC (excitation ~495 nm, emission ~519 nm)

How should researchers validate PITPNA Antibody, FITC conjugated before critical experiments?

Multi-step Validation Protocol:

• Positive and Negative Controls:

  • Use tissues/cells known to express PITPNA (e.g., neuronal cells) as positive controls

  • Use PITPNA-knockout cells or tissues as negative controls

• Peptide Competition Assay:

  • Pre-incubate the antibody with increasing concentrations of the immunizing peptide

  • Observe dose-dependent reduction in signal intensity

• Cross-validation:

  • Compare results with a different PITPNA antibody (different clone or host)

  • Verify consistency in staining patterns

• Knockdown Confirmation:

  • Use siRNA or shRNA against PITPNA to create knockdown models

  • Confirm reduced antibody signal correlates with PITPNA reduction

• Western Blot Correlation:

  • Perform parallel Western blot analysis to confirm specificity and molecular weight

  • Expected molecular weight for human PITPNA is approximately 32 kDa

What controls should be included when using PITPNA Antibody, FITC conjugated?

Essential Controls Table:

How can researchers troubleshoot weak or non-specific signals?

Troubleshooting Guide:

• Weak Signal:

  • Increase antibody concentration

  • Extend incubation time to overnight at 4°C

  • Optimize fixation method (test different fixatives)

  • Enhance antigen retrieval (if using fixed tissues)

  • Ensure sample is properly permeabilized for intracellular targets

• High Background:

  • Increase blocking time and concentration

  • Add 0.1-0.3% Triton X-100 to antibody diluent

  • Use a more stringent washing procedure (more washes, longer duration)

  • Reduce antibody concentration

  • Prepare fresh working dilutions

• Non-specific Binding:

  • Pre-absorb antibody with non-specific proteins

  • Increase blocking with species-specific serum

  • Use alternative blocking agents (BSA, non-fat milk, commercial blockers)

  • Include 0.1% Tween-20 in wash buffers

• Fluorescence Fading:

  • Use anti-fade mounting medium

  • Minimize exposure to light during processing

  • Capture images promptly after staining

  • Store slides at 4°C in the dark

How can PITPNA Antibody, FITC conjugated be used to investigate neurodegenerative disorders?

PITPNA plays critical roles in neuronal function, making it relevant to neurodegenerative research. In mice, loss-of-function mutations of PITPNA cause dose-sensitive phenotypes, including neurological dysfunction, spinocerebellar neurodegeneration, and premature death .

Methodological Approach:

• Comparative Expression Analysis:

  • Use FITC-conjugated PITPNA antibody to compare expression levels in healthy versus diseased brain tissues

  • Quantify differences in subcellular localization and expression intensity

• Co-localization Studies:

  • Combine with markers for neuronal stress (e.g., phosphorylated tau, amyloid-β)

  • Use confocal microscopy to determine spatial relationships between PITPNA and pathological markers

• Cell-type Specific Analysis:

  • Perform dual immunolabeling with neuronal, astrocytic, and microglial markers

  • Assess cell-type specific changes in PITPNA expression in disease models

• Functional Correlation:

  • Correlate PITPNA distribution with phosphoinositide signaling components

  • Analyze relationship between PITPNA localization and neuronal morphology/integrity

What role does PITPNA play in Type 2 diabetes research and how can the antibody be applied?

PITPNA stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate in the trans-Golgi network, which promotes insulin granule maturation . PITPNA expression is markedly reduced in beta-cells of T2D human subjects, suggesting its depletion accompanies beta-cell dysfunction .

Research Applications:

• Beta-cell Analysis:

  • Use FITC-conjugated PITPNA antibody to assess expression in pancreatic islets from normal and diabetic subjects

  • Correlate PITPNA levels with insulin production and secretion metrics

• Insulin Granule Visualization:

  • Co-stain with insulin to examine relationship between PITPNA and insulin granule distribution

  • Analyze granule morphology and maturation in relation to PITPNA expression

• Therapeutic Intervention Assessment:

  • Monitor PITPNA expression changes following anti-diabetic treatments

  • Correlate restoration of PITPNA with improvements in beta-cell function

• Signaling Pathway Analysis:

  • Examine co-localization with PtdIns-4-P and other phosphoinositides

  • Assess relationship between PITPNA, PI3K/Akt pathway components, and insulin secretion machinery

Recent research has shown that restoration of PITPNA in islets of T2D human subjects reverses beta-cell defects, identifying PITPNA as a critical target linked to beta-cell failure in T2D .

How can PITPNA Antibody be used to study muscular dystrophy mechanisms?

PITPNA modulation has shown promising results in muscular dystrophy models. Knockdown of PITPNA in human Duchenne Muscular Dystrophy (DMD) myoblasts increases phosphorylated Akt (pAkt) and fusion index, suggesting therapeutic potential .

Experimental Approach:

• Expression Analysis in Dystrophic Muscle:

  • Use FITC-conjugated PITPNA antibody to compare expression patterns in normal versus dystrophic muscle samples

  • Quantify differences in subcellular distribution

• Signaling Pathway Investigation:

  • Co-stain with pAkt (Ser473) to examine correlation between PITPNA and Akt activation

  • Analyze PTEN levels in relation to PITPNA expression (PITPNA knockdown has been shown to decrease PTEN)

• Myoblast Differentiation Studies:

  • Track PITPNA expression during myoblast fusion and differentiation

  • Correlate changes with fusion index and myotube formation

• Therapeutic Monitoring:

  • Assess PITPNA expression changes in response to potential DMD treatments

  • Use as a biomarker for treatment efficacy

Research has shown that targeted inhibition of PITPNA in dystrophic muscle cells can modulate pAkt expression and improve muscle cell differentiation, positioning PITPNA as a potential DMD therapeutic target .

What are the considerations when using PITPNA Antibody, FITC conjugated in multi-color immunofluorescence experiments?

Critical Considerations:

• Spectral Compatibility:

  • FITC excitation peak: ~495 nm; emission peak: ~519 nm (green)

  • Select secondary fluorophores with minimal spectral overlap (e.g., Cy3, Cy5, Texas Red)

  • Consider using spectral unmixing for closely overlapping fluorophores

• Order of Application:

  • For multiple primary antibodies: Use sequentially rather than simultaneously if from the same host species

  • Apply FITC-conjugated antibodies last in the sequence to minimize photobleaching

• Channel Bleed-through Prevention:

  • Perform single-color controls to establish proper exposure settings

  • Use narrow bandpass filters to minimize signal overlap

  • Adjust acquisition settings to prevent saturation

• Antibody Cross-reactivity:

  • Test antibodies individually before combining

  • Use blocking steps between applications of antibodies from the same species

  • Consider using Fab fragments to block cross-reactivity

• Image Acquisition Strategy:

  • Capture FITC channel images early to minimize photobleaching

  • Use sequential scanning rather than simultaneous acquisition

  • Consider computational post-processing to correct for bleed-through

How do results from PITPNA Antibody, FITC conjugated compare with other detection methods?

Comparative Analysis Table:

What quantitative methods are recommended for analyzing PITPNA expression data?

Quantitative Analysis Approaches:

• Fluorescence Intensity Measurements:

  • Measure mean fluorescence intensity within defined regions of interest

  • Use software like ImageJ, CellProfiler, or commercial imaging platforms

  • Compare relative expression between experimental conditions

• Subcellular Distribution Analysis:

  • Perform co-localization analysis with organelle markers

  • Calculate Pearson's or Mander's coefficients to quantify overlap

  • Use line scan analysis to assess spatial distribution patterns

• Single-Cell Analysis:

  • Segment individual cells using nuclear or membrane markers

  • Measure PITPNA expression on a cell-by-cell basis

  • Create distribution plots to capture population heterogeneity

• Western Blot Correlation:

  • Validate immunofluorescence findings with quantitative Western blot

  • Normalize PITPNA expression to appropriate housekeeping proteins

  • Compare relative expression across experimental conditions

• Flow Cytometry Analysis:

  • Use FITC-conjugated PITPNA antibody for flow cytometry

  • Analyze signal intensity distribution in cell populations

  • Gate on specific cell subsets for targeted analysis

How can researchers account for potential cross-reactivity with other PITP family members?

PITPNA shares significant homology with other phosphatidylinositol transfer proteins, particularly PITPNB. Careful validation is necessary to ensure specificity.

Cross-reactivity Mitigation Strategies:

• Antibody Selection:

  • Choose antibodies raised against unique regions of PITPNA

  • Verify the immunogen sequence does not have high homology with other PITP family members

  • Review manufacturer's cross-reactivity data carefully

• Validation in Knockout/Knockdown Models:

  • Test antibody in PITPNA-specific knockdown models

  • Confirm signal reduction corresponds to degree of knockdown

  • Use CRISPR/Cas9-generated knockout cells as definitive negative controls

• Parallel Detection Approaches:

  • Confirm findings with alternative detection methods (e.g., RNA detection via FISH)

  • Use isoform-specific PCR to correlate protein expression with mRNA levels

  • Consider mass spectrometry validation for complex samples

• Comparative Expression Analysis:

  • Analyze expression in tissues with known differential expression of PITP family members

  • PITPNA is predominantly expressed in neurons, while other family members may have different tissue distribution

  • Research has shown that PITPNB expression remains unchanged in beta-cells of T2D human donors while PITPNA is reduced

• Peptide Competition:

  • Perform pre-absorption with specific peptides from different PITP family members

  • Only PITPNA-specific peptides should block antibody binding