PSEN2 Antibody, FITC conjugated

What is PSEN2 and why is it important in neurodegenerative disease research?

PSEN2 (presenilin 2) is one of three proteins that, when mutated, causes early onset familial Alzheimer disease (FAD). It functions as a probable catalytic 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) . In addition to its well-documented role in the γ-secretase complex, PSEN2 is endowed with γ-secretase-independent functions in distinct cell signaling pathways, such as modulating intracellular Ca²⁺ homeostasis . Research on PSEN2 is critical for understanding the pathological mechanisms underlying Alzheimer's disease, as mutations in this protein can influence autophagy, calcium signaling, and amyloid processing.

What are the main applications for PSEN2 antibodies in experimental research?

PSEN2 antibodies are utilized across multiple experimental techniques including:

• Western Blotting (WB): For protein detection and quantification in cell/tissue lysates

• Immunohistochemistry (IHC): For localization studies in tissue sections

• Immunofluorescence (IF): For subcellular localization studies

• ELISA: For quantitative detection in solution

• FACS (Flow Cytometry): For cell-surface or intracellular detection

The choice of application depends on research objectives, with WB commonly used for expression analysis, IHC/IF for localization studies, and ELISA for quantitative measurements of PSEN2 levels in biological samples.

What does FITC conjugation provide in PSEN2 antibody applications?

FITC (Fluorescein isothiocyanate) conjugation enables direct visualization of PSEN2 protein through fluorescence detection without requiring secondary antibody incubation. This conjugation provides:

• Direct detection capability in fluorescence microscopy and flow cytometry

• Excitation maximum around 495 nm and emission maximum around 519 nm (green fluorescence)

• Reduced protocol time by eliminating secondary antibody incubation steps

• Potential for multiplexing with other fluorophores in co-localization studies

When using FITC-conjugated PSEN2 antibodies, researchers should protect the reagent from light exposure to prevent photobleaching and maintain optimal storage conditions (-20°C) to preserve fluorescence activity .

What is the optimal protocol for using PSEN2 Antibody, FITC conjugated in immunofluorescence applications?

For optimal immunofluorescence results with FITC-conjugated PSEN2 antibody:

• Sample preparation:

  • Fix cells using 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.1-0.3% Triton X-100 in PBS (10 minutes)

  • Block with 1-5% BSA or normal serum in PBS (1 hour)

• Antibody incubation:

  • Dilute FITC-conjugated PSEN2 antibody to appropriate concentration (typically 1:150 for immunofluorescence)

  • Incubate in humidified chamber at 4°C overnight or 1-2 hours at room temperature

  • Protect from light during all steps

• Washing and mounting:

  • Wash 3x with PBS to remove unbound antibody

  • Counterstain nucleus with DAPI if desired

  • Mount with anti-fade mounting medium

• Imaging considerations:

  • Use appropriate filter set for FITC (excitation ~495 nm, emission ~519 nm)

  • Minimize exposure time to prevent photobleaching

  • Include negative controls (secondary antibody only) and positive controls

The dilution should be optimized for each experimental system, with the recommended starting dilution being 1:150 for immunofluorescence applications .

How should PSEN2 Antibody, FITC conjugated be stored and handled to maintain optimal performance?

Proper storage and handling are crucial for maintaining antibody performance:

Storage recommendations:

• Store at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles by preparing working aliquots

• Keep protected from light to prevent photobleaching of the FITC fluorophore

• Most preparations remain stable for at least one year when stored properly

Handling guidelines:

• Upon receipt, either store intact at -20°C or prepare working aliquots

• Thaw aliquots completely before use and mix gently by inversion (avoid vortexing)

• If in liquid form, the antibody is typically supplied in a buffer containing stabilizers (e.g., 50% glycerol, 0.01M PBS, pH 7.4)

• Some preparations may contain preservatives like 0.03% Proclin 300 or sodium azide

Reconstitution (if lyophilized):

• Add specified volume of distilled/deionized water or recommended buffer

• Allow complete reconstitution (15-20 minutes at room temperature)

• For some preparations, add 0.2 ml of distilled water to yield a concentration of 500 μg/ml

Always refer to the specific product documentation for exact storage and handling recommendations, as formulations may vary between manufacturers.

How can PSEN2 Antibody, FITC conjugated be utilized in studying Alzheimer's disease mechanisms involving mutant PSEN2?

FITC-conjugated PSEN2 antibodies provide valuable tools for investigating the molecular mechanisms of Alzheimer's disease, particularly in contexts involving mutant PSEN2:

• Subcellular localization studies:

  • Visualize altered localization patterns of mutant PSEN2 proteins compared to wild-type

  • Examine co-localization with organelle markers to determine subcellular distribution changes

  • Quantify differences in nuclear, ER, or Golgi localization between wild-type and mutant forms

• Protein-protein interaction analyses:

  • Combine with antibodies against other γ-secretase components to study complex formation

  • Investigate interactions with calcium signaling proteins to explore γ-secretase-independent functions

  • Examine co-localization with autophagy markers to assess impact on degradative pathways

• iPSC-derived cell models:

  • Recent research has successfully differentiated iPSC-derived microglia and astrocytes from patients harboring PSEN2 (N141I) mutations

  • FITC-conjugated PSEN2 antibodies can be used to characterize these cellular models

  • These models revealed that PSEN2 (N141I)-mutant astrocytes and microglia-like cells present with a 'primed' phenotype characterized by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion, and altered Aβ₄₂ production and phagocytosis

• Autophagy studies:

  • FAD-PSEN2 has been shown to impair autophagy by blocking the degradative flux at the autophagosome-lysosome fusion step

  • FITC-conjugated PSEN2 antibodies can help visualize PSEN2 localization relative to autophagy markers

  • Combined with markers like LC3 and SQSTM1/p62, these antibodies enable assessment of PSEN2's impact on autophagy dynamics

These approaches provide mechanistic insights into how PSEN2 mutations contribute to Alzheimer's disease pathogenesis beyond simple amyloid processing alterations.

What are the key considerations when choosing between different epitope-specific PSEN2 antibodies?

Epitope selection is critical when studying PSEN2, as different antibodies target distinct regions of the protein with important functional implications:

• N-terminal epitopes (amino acids 1-50, 32-65, 33-48, 39-51):

  • Suitable for detecting full-length PSEN2

  • Several commercial antibodies target this region

  • Important for identifying proteins before endoproteolytic processing

  • May not detect C-terminal fragments after cleavage

• C-terminal epitopes (e.g., aa 319-334):

  • Detect C-terminal fragments after endoproteolytic processing

  • Useful for studying PSEN2 processing and turnover

  • May help distinguish between different cleavage products

• Mid-region epitopes (e.g., aa 101-150):

  • Can provide detection of multiple forms of the protein

  • May be useful for general PSEN2 detection regardless of processing state

• Consideration of PSEN2 mutations:

  • For studies involving specific mutations (e.g., N141I, T122R), ensure the antibody epitope does not overlap with or is affected by the mutation site

  • Some mutations may alter protein conformation and affect antibody binding affinity

• Species cross-reactivity:

  • Different epitopes show varying degrees of conservation across species

  • Some antibodies demonstrate broad reactivity across human, mouse, rat, and other species

  • Others have more limited species reactivity

  • Certain N-terminal epitopes differ between human and rodent sequences by two amino acids

When selecting a PSEN2 antibody, researchers should consider their specific experimental questions and whether they need to detect full-length protein, specific fragments, or particular mutant forms.

What are common issues encountered when using PSEN2 Antibody, FITC conjugated, and how can they be resolved?

Researchers may encounter several challenges when working with FITC-conjugated PSEN2 antibodies:

Problem: Weak or no signal

• Possible causes:

  • Insufficient antibody concentration

  • Low target protein expression

  • Photobleaching of FITC conjugate

  • Inadequate permeabilization

  • Improper sample fixation

• Solutions:

  • Increase antibody concentration (try 1:100 dilution if 1:150 was insufficient)

  • Extend incubation time (overnight at 4°C)

  • Protect from light during all procedures

  • Optimize permeabilization conditions (try increasing Triton X-100 to 0.3-0.5%)

  • Try different fixation methods (paraformaldehyde vs. methanol)

Problem: High background signal

• Possible causes:

  • Excessive antibody concentration

  • Insufficient blocking

  • Inadequate washing

  • Autofluorescence of sample

• Solutions:

  • Decrease antibody concentration

  • Extend blocking time or increase blocking agent concentration (5% BSA)

  • Increase number and duration of wash steps

  • Include 0.05-0.1% Tween-20 in wash buffer

  • For tissue sections, treat with autofluorescence reducers

Problem: Non-specific binding

• Possible causes:

  • Cross-reactivity with other proteins

  • Fc receptor binding in immune cells

  • Non-specific binding to dead or damaged cells

• Solutions:

  • Include appropriate negative controls

  • Preincubate samples with Fc receptor blocking reagent

  • Perform viability assays and exclude dead cells from analysis

  • Consider using a more specific antibody targeting a different epitope

Problem: Unexpected molecular weight in Western blot

• Possible causes:

  • Post-translational modifications

  • Protein cleavage/processing

  • Incorrect sample preparation

• Solutions:

  • PSEN2 is often detected at 55-60 kDa though calculated at 50 kDa

  • PSEN2 undergoes endoproteolytic processing into N-terminal (34 kDa) and C-terminal (23 kDa) fragments

  • Use appropriate positive controls to confirm expected banding pattern

  • Consider different sample preparation methods to preserve protein integrity

How can researchers optimize dual-labeling experiments involving FITC-conjugated PSEN2 antibodies?

Optimizing dual-labeling experiments requires careful consideration of fluorophore compatibility and experimental design:

• Fluorophore selection for co-staining:

  • Choose secondary fluorophores with minimal spectral overlap with FITC (green)

  • Recommended partners: red fluorophores (e.g., Cy3, Texas Red, Alexa Fluor 594)

  • Avoid yellow-green fluorophores like PE that may have spectral overlap

  • Far-red fluorophores (e.g., Cy5, Alexa Fluor 647) also work well with FITC

• Sequential vs. simultaneous staining:

  • Sequential staining (generally preferred):

    • Complete FITC-PSEN2 staining protocol, then perform secondary antibody staining

    • Minimizes cross-reactivity between antibodies

    • May require additional blocking step between staining sequences

  • Simultaneous staining:

    • Incubate with FITC-PSEN2 antibody and unconjugated primary antibody together

    • Add appropriate secondary antibody for the unconjugated primary

    • More time-efficient but higher risk of cross-reactivity

• Controls for dual-labeling experiments:

  • Single-stained controls (each antibody alone)

  • Secondary-only controls

  • Isotype controls

  • Absorption controls (pre-incubation with blocking peptide)

• Image acquisition optimization:

  • Capture single-fluorophore images sequentially rather than simultaneously

  • Apply appropriate compensation if using flow cytometry

  • Consider spectral unmixing for confocal microscopy with significant overlap

  • Begin with the longest wavelength fluorophore when acquiring images to minimize photobleaching of FITC

• Co-localization analysis:

  • Use appropriate software tools (ImageJ with coloc2, CellProfiler)

  • Apply rigorous statistical analysis (Pearson's correlation, Manders' coefficients)

  • Establish threshold values based on control samples

  • Consider 3D analysis for volumetric co-localization assessment

By carefully optimizing these parameters, researchers can achieve reliable dual-labeling results when studying PSEN2 in relation to other proteins of interest.

How are PSEN2 antibodies being used to study autophagy impairment in Alzheimer's disease models?

PSEN2 antibodies are instrumental in revealing the relationship between mutated PSEN2 and autophagy dysfunction in Alzheimer's disease:

• Autophagosome-lysosome fusion studies:

  • Research demonstrates that FAD-PSEN2 impairs autophagy by blocking the degradative flux at the autophagosome-lysosome fusion step

  • This blockage does not depend on altered lysosomal functionality but rather on decreased recruitment of the small GTPase RAB7 to autophagosomes

  • PSEN2 antibodies help visualize the localization of PSEN2 relative to autophagy markers LC3-II and SQSTM1/p62

• Quantitative assessment of autophagy markers:

  • Western blot analysis using PSEN2 antibodies alongside autophagy markers reveals:

    • Significant increase (almost 2-fold) in lipidated LC3-II in cells expressing PSEN2 T122R compared to controls

    • Increased autophagy receptor SQSTM1/p62 levels in PSEN2 T122R-expressing cells

    • These changes indicate autophagy impairment rather than increased autophagic flux

• Colocalization analysis with autophagy components:

  • Fluorescently-labeled PSEN2 antibodies enable visualization of:

    • PSEN2 localization relative to autophagosome markers

    • Changes in autophagosome size and number (significantly larger in FAD-PSEN2 expressing cells)

    • Differential recruitment of RAB7 to autophagosomes in wild-type versus mutant PSEN2-expressing cells

• Calcium signaling connection:

  • PSEN2 antibodies help demonstrate that FAD-PSEN2's action on autophagy is unrelated to its γ-secretase activity

  • Instead, it depends on PSEN2's ability to partially deplete ER Ca²⁺ content, reducing cytosolic Ca²⁺ response upon IP3-linked cell stimulations

  • This provides mechanistic insight into how PSEN2 mutations impact cellular homeostasis beyond amyloid processing

These applications of PSEN2 antibodies have revealed a novel mechanism by which FAD-linked presenilins alter the degradative process, reinforcing the view of a causative role for dysfunctional quality control pathways in AD neurodegeneration.

What insights have been gained from studying PSEN2 in iPSC-derived glial cells using fluorescent antibody techniques?

Recent research utilizing iPSC-derived glial cells has provided novel insights into PSEN2's role in non-neuronal cells in Alzheimer's disease:

• Establishment of novel cellular models:

  • For the first time, researchers have successfully differentiated iPSC-derived astrocytes and microglia-like cells from patients harboring a PSEN2 (N141I) mutation

  • These models enable investigation of PSEN2's role in glial cells, expanding beyond the traditional neuronal focus

• Altered glial morphology and function:

  • FITC-conjugated antibodies and related fluorescent techniques revealed:

    • PSEN2 (N141I)-mutant astrocytes and microglia-like cells present with a 'primed' phenotype

    • Reduced morphological complexity in mutant glial cells

    • Exaggerated pro-inflammatory cytokine secretion patterns

    • Altered Aβ₄₂ production and phagocytic capabilities

• Mechanistic insights:

  • Fluorescent antibody techniques helped demonstrate:

    • Changes in protein expression profiles in PSEN2 mutant glial cells

    • Alterations in inflammatory signaling pathways

    • Differences in amyloid processing and clearance capabilities

    • Cell-type specific responses to PSEN2 mutations

• Implications for therapeutic development:

  • These studies suggest:

    • Glial-specific interventions may be necessary alongside neuronal-targeted therapies

    • Anti-inflammatory approaches might benefit patients with PSEN2 mutations

    • The need for cell-type specific drug screening using these iPSC-derived models

    • Potential for personalized medicine approaches based on specific mutations

This research expands our understanding of PSEN2 beyond neurons and γ-secretase activity, highlighting its importance in glial cell function and neuroinflammation in Alzheimer's disease pathogenesis.

How do FITC-conjugated PSEN2 antibodies compare with other detection methods for studying presenilin function?

Different detection methods offer various advantages and limitations when studying PSEN2:

When selecting between these methods, researchers should consider:

• Research question specificity:

  • For localization studies, FITC-conjugated antibodies offer direct visualization

  • For protein-protein interactions, proximity ligation assays or co-IP with unconjugated antibodies may be superior

  • For quantitative expression analysis, WB with unconjugated antibodies typically provides better sensitivity

• Target abundance considerations:

  • For low-abundance PSEN2 detection, signal amplification with unconjugated primary + secondary approach may be preferred

  • For high-abundance targets, direct FITC conjugation offers simplified workflows

• Multiplexing requirements:

  • When studying PSEN2 alongside multiple other proteins, consider the entire fluorophore panel

  • FITC occupies the green channel, so plan additional markers accordingly

Each method has its place in comprehensive PSEN2 research, with the optimal approach depending on specific experimental objectives and available resources.