PSENEN Antibody, FITC conjugated

What is PSENEN and why are FITC-conjugated antibodies against it useful in research?

PSENEN (also known as PEN-2 or Presenilin enhancer protein 2) is an essential subunit of the gamma-secretase complex, an endoprotease complex that catalyzes the intramembrane cleavage of integral membrane proteins such as Notch receptors and APP (beta-amyloid precursor protein) . FITC-conjugated PSENEN antibodies enable direct visualization of this protein in various experimental applications without requiring secondary antibody incubation. The conjugated fluorophore (FITC) has an excitation wavelength of 488 nm and emission at approximately 535 nm, allowing compatibility with standard flow cytometry equipment and fluorescence microscopy setups . These antibodies are particularly valuable for multicolor immunofluorescence experiments where PSENEN can be co-localized with other proteins of the gamma-secretase complex such as PSEN1, nicastrin, or APH1 .

What applications are most suitable for PSENEN antibody, FITC conjugated?

The FITC-conjugated PSENEN antibody has been validated for multiple applications including:

• ELISA: For quantitative measurement of PSENEN in cell lysates and tissue homogenates

• Flow cytometry: For analyzing PSENEN expression in cell populations with recommended dilutions of 1:50-1:100

• Immunocytochemistry (ICC): For cellular localization studies with recommended dilutions of 1:50-1:200

• Western blotting: For protein expression analysis with recommended dilutions of 1:500-1:2,000

• Immunohistochemistry (IHC): For tissue localization with recommended dilutions of 1:50-1:200

When designing experiments, researchers should consider that PSENEN is a small protein (12 kDa calculated molecular weight) that functions primarily as part of the gamma-secretase complex . Its detection may require specific sample preparation techniques to maintain the integrity of membrane protein complexes.

How should samples be prepared for optimal PSENEN detection using FITC-conjugated antibodies?

For optimal detection of PSENEN using FITC-conjugated antibodies, sample preparation should account for both the membrane-bound nature of the protein and the properties of the fluorophore:

• For flow cytometry: Cells should be fixed with 4% paraformaldehyde and permeabilized with a mild detergent (0.1% Triton X-100 or 0.1% saponin). Pre-incubation with 5% fetal bovine serum for 10 minutes helps block nonspecific binding. Cells should be incubated with the antibody for 1 hour at 4°C .

• For immunohistochemistry: Tissues should be fixed in 4% paraformaldehyde, embedded in paraffin or frozen, and sectioned. Antigen retrieval may be necessary for optimal staining. Autofluorescence quenching is recommended for tissue samples to improve signal-to-noise ratio .

• For western blotting: Standard membrane protein extraction using CHAPSO detergent (which preserves gamma-secretase complex integrity) is recommended. Blue native PAGE can be used to assess complex integrity .

• Storage considerations: The antibody solution should be stored at -20°C for long-term storage and at 4°C for up to one month for frequent use. Repeated freeze-thaw cycles should be avoided .

How can PSENEN antibody, FITC conjugated be used to study the gamma-secretase complex assembly and stoichiometry?

The gamma-secretase complex consists of four essential proteins—PSEN, nicastrin (NCSTN), anterior pharynx defective 1 (APH1), and PSENEN—in a 1:1:1:1 stoichiometry . FITC-conjugated PSENEN antibodies can be used in combination with other fluorescently labeled antibodies against different complex components to study assembly dynamics and stoichiometry through several advanced approaches:

• Super-resolution microscopy: This technique can resolve the spatial distribution of PSENEN relative to other gamma-secretase components with nanometer precision. Recent studies using structured illumination microscopy (SIM) revealed that gamma-secretase complexes exist as distinct spots at the plasma membrane with nearest-neighbor distances that reflect their association .

• Co-immunoprecipitation with fluorescence detection: FITC-labeled PSENEN antibodies can be used to pull down the complex, followed by fluorescence-based detection of co-precipitated components, providing quantitative information about complex stoichiometry .

• Fluorescence resonance energy transfer (FRET): By pairing FITC-labeled PSENEN antibodies with antibodies against other complex components conjugated to compatible acceptor fluorophores, researchers can measure the proximity between different subunits of the gamma-secretase complex in living cells .

• Fluorescence correlation spectroscopy (FCS): This technique can measure the diffusion characteristics of FITC-labeled complexes, providing information about the size and composition of the gamma-secretase complex in different cellular compartments.

What approaches can be used to validate PSENEN antibody specificity in experimental systems?

Validating antibody specificity is crucial for obtaining reliable research results. For PSENEN antibody, FITC conjugated, several validation approaches are recommended:

• Genetic knockout controls: Use of PSENEN knockout cell lines (such as CRISPR/Cas9-engineered cells) as negative controls is the gold standard for antibody validation. The absence of signal in knockout cells confirms specificity .

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining if the antibody is truly specific. Some manufacturers offer blocking peptides that can be purchased separately .

• Correlation with alternative detection methods: Results from FITC-conjugated antibody staining should correlate with mRNA expression levels measured by RT-PCR or with protein levels detected by mass spectrometry.

• Cross-validation with multiple antibodies: Using multiple antibodies targeting different epitopes of PSENEN helps confirm specificity of the observed signals.

• Western blot analysis: A single band at the expected molecular weight (approximately 12 kDa for PSENEN) supports antibody specificity .

• Tandem affinity purification: This technique can be used to identify potential cross-reactivity by identifying all proteins precipitated by the antibody, as demonstrated in studies of PSENEN-interacting proteins .

How can PSENEN antibody, FITC conjugated be employed in studies of Alzheimer's disease pathogenesis?

PSENEN, as part of the gamma-secretase complex, plays a critical role in the production of amyloid-beta (Aβ) peptides implicated in Alzheimer's disease pathogenesis. FITC-conjugated PSENEN antibodies can be used in several advanced approaches for AD research:

• Cell-based assays for gamma-secretase modulator screening: FITC-conjugated PSENEN antibodies can be used to monitor changes in complex formation or localization in response to potential therapeutic compounds that modulate gamma-secretase activity .

• Investigation of PSEN1/PSEN2 selectivity: Recent studies have identified selective inhibitors of PSEN1-gamma-secretase complex that spare PSEN2 complexes. FITC-conjugated PSENEN antibodies can help elucidate the differential composition and localization of PSEN1 versus PSEN2 containing complexes in various cellular contexts .

• Analysis of familial Alzheimer's disease (FAD) mutations: The effects of FAD mutations in PSEN1 or PSEN2 on complex assembly and cellular localization can be studied using colocalization analysis with FITC-conjugated PSENEN antibodies and other labeled components .

• Investigation of autophagy-lysosome pathway connections: Recent research has identified converging roles of PSENEN/PEN2 and CLN3 in the autophagy-lysosome system. FITC-conjugated PSENEN antibodies can help map the interactions between these pathways in neurodegenerative disease models .

• Study of cellular mechanisms in cholinergic-like neurons: FITC-conjugated antibodies can be used to investigate PSENEN expression and localization in models of FAD, such as those carrying the PSEN1 E280A mutation, providing insights into pathological mechanisms in specific neuronal populations .

How can background fluorescence be minimized when using PSENEN antibody, FITC conjugated?

Background fluorescence can significantly impact the quality of data obtained with FITC-conjugated antibodies. Researchers can employ several strategies to minimize background:

• Optimized blocking: Use 5% normal serum from the same species as the secondary antibody for at least 30 minutes before antibody incubation. For flow cytometry applications, pre-incubation with 5% fetal bovine serum for 10 minutes has been shown to effectively block nonspecific binding .

• Autofluorescence quenching: For tissue sections, treatment with 0.1% Sudan Black B in 70% ethanol for 20 minutes can significantly reduce autofluorescence from lipofuscin and other endogenous fluorophores.

• Antibody titration: Perform dilution series experiments to determine the optimal antibody concentration that provides sufficient specific signal while minimizing background. Starting dilutions of 1:50-1:200 for ICC/IHC and 1:500-1:2,000 for western blotting are recommended .

• Buffer optimization: The buffer composition (PBS with 0.03% Proclin 300, 50% Glycerol, pH 7.4) is optimized for maintaining antibody stability and specificity. Modifying buffer components may be necessary for specific applications .

• Sample preparation: Freshly prepared samples generally yield better results with lower background. For fixed samples, complete permeabilization is essential for detecting intracellular PSENEN, but excessive permeabilization can increase nonspecific binding.

• Negative controls: Always include isotype controls (non-specific IgG-FITC) and unstained samples to establish background levels and set appropriate gates for flow cytometry analysis.

What are the potential cross-reactivity concerns with PSENEN antibody, FITC conjugated?

Understanding potential cross-reactivity is essential for accurate interpretation of experimental results. For PSENEN antibodies, several considerations should be addressed:

• Sequence homology: PSENEN shares structural features with other small transmembrane proteins. Antibodies raised against synthetic peptides corresponding to C-terminal human PEN2 may cross-react with structurally similar proteins .

• Species cross-reactivity: While many PSENEN antibodies are designed to recognize human protein, cross-reactivity with mouse and rat homologs varies between products. Researchers should verify species reactivity for their specific application, particularly in comparative studies using multiple model organisms .

• Complex integrity: PSENEN exists primarily in complex with other gamma-secretase components. Antibody accessibility to epitopes may be affected by complex formation or conformation, potentially leading to differential detection of free versus complexed PSENEN.

• Validation in multiple systems: Researchers should validate antibody specificity in their specific experimental system using approaches outlined in question 2.2, particularly when working with new cell types or tissues.

• Epitope considerations: Antibodies targeting different regions of PSENEN may yield different results due to epitope accessibility or post-translational modifications. Information about the immunogen (e.g., "Peptide sequence from Human Gamma-secretase subunit PEN-2 protein (39-57AA)") helps assess potential recognition sites .

How does sample fixation affect PSENEN antibody, FITC conjugated binding and fluorescence signal?

Sample fixation is a critical step that can significantly impact antibody binding and fluorescence signal quality. For PSENEN antibody, FITC conjugated:

How does PSENEN antibody, FITC conjugated compare to other fluorophore conjugates for multi-color imaging applications?

When designing multi-color imaging experiments, the choice of fluorophore conjugation impacts experimental design and data quality:

• Spectral considerations: FITC (excitation 488 nm, emission 535 nm) occupies the green channel in most imaging systems . This positioning has implications for multi-color experiment design:

  • Advantages: FITC is compatible with most standard fluorescence microscopes and flow cytometers

  • Limitations: FITC has significant spectral overlap with GFP, limiting its use in GFP-expressing systems

• Alternative fluorophores: Compared to other conjugates, FITC has specific characteristics:

  • FITC vs. PE (phycoerythrin): PE provides brighter signal but is larger and may affect antibody binding kinetics in some applications

  • FITC vs. Alexa Fluor 488: Alexa Fluor 488 offers greater photostability and less pH sensitivity but is typically more expensive

  • FITC vs. PerCP-Cy5.5: PerCP-Cy5.5 emits in the far-red spectrum, allowing greater separation from autofluorescence and compatibility with GFP-based systems

• Photobleaching considerations: FITC is more susceptible to photobleaching than newer fluorophores like Alexa Fluor dyes. For long-term imaging or repeated scanning, alternative conjugations may be preferable.

• Multi-color combinations: For co-localization studies with other gamma-secretase components, complementary fluorophore pairs are recommended:

  • PSENEN (FITC) + PSEN1 (far-red fluorophores like Cy5 or Alexa 647)

  • PSENEN (FITC) + Nicastrin (red fluorophores like PE or Alexa 594)

• Super-resolution microscopy compatibility: For techniques like STORM or PALM, Alexa Fluor dyes or other photoswitchable fluorophores may provide superior performance compared to FITC.

What are the optimal approaches for co-immunoprecipitation studies involving PSENEN antibody, FITC conjugated?

Co-immunoprecipitation (co-IP) is valuable for studying protein-protein interactions involving PSENEN within the gamma-secretase complex:

• FITC antibody immobilization: FITC-conjugated antibodies can be immobilized on anti-FITC beads or anti-fluorescein matrices for immunoprecipitation. Alternatively, anti-IgG beads can capture the PSENEN antibody-antigen complex directly.

• Detergent selection: The choice of detergent is critical for maintaining gamma-secretase complex integrity:

  • CHAPSO (0.5-1%) preserves gamma-secretase complex integrity and is preferred for studying intact complexes

  • n-Dodecyl β-D-maltoside (DDM, 0.5%) has been successfully used to maintain high molecular weight complexes while extracting membrane proteins

  • Triton X-100 (1%) has been used for general protein extraction but may disrupt some protein-protein interactions

• Validation approaches: Successful co-IP can be validated by:

  • Western blotting for expected complex components (PSEN1-NTF, PSEN1-CTF, Nicastrin)

  • Direct fluorescence measurement of FITC signal in precipitated complexes

  • Mass spectrometry analysis of co-precipitated proteins

• Reciprocal precipitation: For thorough validation, reciprocal co-IP using antibodies against other complex components (e.g., GFP-PSEN1) should co-precipitate PSENEN-FITC in comparable ratios to input samples .

• Quantitative analysis: Blue native PAGE of DDM-extracted membrane preparations can confirm complex integrity before and after immunoprecipitation .

• Advanced applications: Tandem affinity purification using PSENEN with C-terminal TAP-Tag followed by mass spectrometry has been used to identify novel interaction partners like CLN3, expanding our understanding of PSENEN functions beyond the gamma-secretase complex .

How can super-resolution microscopy enhance the application of PSENEN antibody, FITC conjugated in studies of membrane protein dynamics?

Super-resolution microscopy overcomes the diffraction limit of conventional fluorescence microscopy, offering powerful approaches for studying PSENEN distribution and dynamics:

• Structured Illumination Microscopy (SIM): This technique achieves resolution of ~100 nm and has been successfully used to visualize gamma-secretase components at the plasma membrane. SIM reveals that GFP-PSEN1 spots overlap with or adjoin NCT-SNAP-SiR spots, supporting the biochemical prediction of a 1:1 ratio in gamma-secretase complexes .

• Nearest-neighbor analysis: This analytical approach measures the distance between centroid coordinates of fluorescent spots, revealing associations between different gamma-secretase components. For PSENEN studies, this technique can determine spatial relationships with other complex components .

• Single-molecule localization microscopy (STORM/PALM): These techniques achieve resolution of ~20 nm but typically require photoswitchable fluorophores. While FITC itself is not ideal for STORM/PALM, PSENEN antibodies conjugated to appropriate dyes could be used to study nanoscale organization of gamma-secretase complexes.

• Live-cell super-resolution techniques: Approaches like STED (Stimulated Emission Depletion) microscopy can be combined with FITC-conjugated antibodies against surface epitopes to study the dynamics of cell-surface PSENEN in living cells, though photobleaching may limit extended imaging.

• Multi-color super-resolution: By combining FITC-conjugated PSENEN antibodies with other spectrally distinct fluorophores, researchers can map spatial relationships between different components of the gamma-secretase complex or between PSENEN and potential interaction partners like CLN3 .

• Membrane sheet preparation: The preparation of plasma membrane sheets, combined with super-resolution microscopy, has proven effective for studying the distribution of gamma-secretase complexes. This approach minimizes background from intracellular pools and provides excellent signal-to-noise ratio for surface PSENEN visualization .

How might PSENEN antibody, FITC conjugated contribute to understanding the differential roles of PSEN1 versus PSEN2 complexes in gamma-secretase function?

Recent research has revealed that gamma-secretase complexes containing PSEN1 versus PSEN2 have distinct properties and selectivity profiles. FITC-conjugated PSENEN antibodies could contribute to this emerging field in several ways:

• Differential localization studies: FITC-conjugated PSENEN antibodies could be used in combination with PSEN1- and PSEN2-specific antibodies to map the subcellular distribution of different complex types. This approach could reveal whether PSENEN association with PSEN1 versus PSEN2 influences trafficking to specific compartments .

• Drug selectivity mechanisms: Compounds like MRK-560 show selectivity for PSEN1 complexes over PSEN2 complexes. FITC-conjugated PSENEN antibodies could help visualize whether these compounds alter the distribution, assembly, or stability of different complex types .

• Conformational dynamics: Different gamma-secretase complexes may adopt distinct conformations during substrate processing. Advanced fluorescence techniques like FRET using FITC-PSENEN antibodies paired with other labeled components could reveal conformational differences between PSEN1 and PSEN2 complexes.

• Quantitative stoichiometry: Flow cytometry and quantitative microscopy with FITC-conjugated PSENEN antibodies could determine whether the stoichiometry of PSENEN differs between PSEN1 and PSEN2 complexes in different cellular contexts.

• Interaction with substrate trafficking: FITC-conjugated PSENEN antibodies could be used to study how substrate proteins like APP and Notch interact with different gamma-secretase complex types, potentially revealing mechanisms underlying the differential processing observed with different complexes .

What role might PSENEN antibody, FITC conjugated play in investigating connections between gamma-secretase activity and autophagy-lysosome pathways?

Recent research has identified unexpected connections between PSENEN/PEN2 and the autophagy-lysosome system, opening new research directions:

• Co-localization with autophagy markers: FITC-conjugated PSENEN antibodies can be used to study co-localization with autophagy markers like LC3 and LAMP1, especially in the context of neurodegenerative diseases. Studies have already shown PSENEN localization in LAMP1-positive organelles .

• Study of PSENEN-CLN3 interactions: The identification of CLN3 as a PSENEN interaction partner suggests connections to the autophagy-lysosome system. FITC-conjugated PSENEN antibodies could be used to map this interaction in different cellular compartments and disease states .

• Gamma-secretase independent functions: PSENEN may have functions independent of its role in the gamma-secretase complex, particularly in autophagy regulation. FITC-conjugated antibodies could help distinguish between pools of PSENEN associated with gamma-secretase versus other protein complexes.

• Trafficking studies: Live-cell imaging with surface-labeled FITC-PSENEN antibodies could track the internalization and trafficking of surface gamma-secretase complexes to lysosomes, providing insights into their turnover and potential roles in these compartments.

• Therapeutic implications: Understanding the dual role of PSENEN in proteolysis and autophagy could reveal new therapeutic approaches for diseases like Alzheimer's. FITC-conjugated antibodies could be used to screen compounds that selectively modulate one function without affecting the other.

How can PSENEN antibody, FITC conjugated be employed in developing cellular models for therapeutic screening in neurodegenerative diseases?

The development of relevant cellular models is crucial for therapeutic discovery in neurodegenerative diseases. FITC-conjugated PSENEN antibodies can contribute to these efforts:

• High-content screening platforms: FITC-conjugated PSENEN antibodies enable high-throughput, automated imaging to assess gamma-secretase complex assembly, localization, and function in response to potential therapeutic compounds. This approach can be integrated into phenotypic screening workflows.

• Patient-derived cell models: FITC-conjugated PSENEN antibodies can be used to characterize gamma-secretase complexes in patient-derived cells carrying disease mutations, such as the PSEN1 E280A mutation associated with familial Alzheimer's disease . This allows correlation of complex dynamics with disease phenotypes.

• CRISPR-engineered model systems: Complementary to patient cells, CRISPR-engineered model systems with specific mutations in gamma-secretase components provide valuable tools for understanding disease mechanisms. FITC-conjugated PSENEN antibodies can verify complex assembly and localization in these models .

• Validation of therapeutic mechanisms: For compounds designed to modulate gamma-secretase activity, FITC-conjugated PSENEN antibodies can help determine whether the mechanism involves altering complex assembly, stability, or localization.

• Biomarker development: Changes in PSENEN expression, localization, or complex formation might serve as cellular biomarkers for disease progression or treatment response. FITC-conjugated antibodies enable quantitative assessment of these parameters in various model systems.

• Combination with functional readouts: Linking PSENEN visualization to functional readouts such as substrate processing (APP-CTF levels) or downstream signaling (GSK3β phosphorylation) provides a more comprehensive view of potential therapeutic effects .