SHISA9 Antibody, FITC conjugated

What is SHISA9 and why is it relevant for neuroscience research?

SHISA9 is a type-I transmembrane protein containing a C-terminal PDZ domain that interacts with cytosolic proteins in the postsynaptic density . Research demonstrates that SHISA9 modulates AMPA receptor function through interactions with PDZ domain-containing proteins, affecting glutamatergic synaptic transmission . This protein is particularly significant because it influences AMPA receptor kinetics and synaptic facilitation, critical processes for synaptic plasticity and network activity in the hippocampus .

How does SHISA9 function at the molecular level?

SHISA9 functions by interacting with AMPA receptors and several PDZ domain-containing proteins in the postsynaptic density, including PSD95, PSD93, PICK1, GRIP1, and Lin7b through its C-terminal EVTV motif . These interactions anchor SHISA9 at the postsynaptic membrane and influence AMPA receptor-mediated synaptic currents . Research has demonstrated that when these interactions are disrupted (using C-terminal mimetic peptides), glutamatergic AMPA receptor-mediated synaptic currents show faster decay time and reduced paired-pulse facilitation, indicating that SHISA9 modulates both the kinetics and recovery from desensitization of synaptic AMPARs .

What experimental approaches can detect native SHISA9 protein in neural samples?

Multiple experimental approaches can detect native SHISA9 protein:

• Co-immunoprecipitation from brain tissue using anti-SHISA9 antibodies (as demonstrated in the hippocampus and cortex) .

• Western blotting of brain fractions to determine region-specific expression levels.

• Subcellular fractionation focusing on postsynaptic density enriched fractions.

• Immunohistochemistry/immunofluorescence using FITC-conjugated SHISA9 antibodies.

• Proximity ligation assays to detect SHISA9 interactions with binding partners.

When isolating SHISA9 from brain tissue, special solubilization conditions are necessary due to the densely packed protein structure of the postsynaptic density. Research has successfully used buffers containing DDM (dodecyl maltoside) detergent to solubilize SHISA9-containing complexes while maintaining protein-protein interactions .

What advantages does a FITC-conjugated SHISA9 antibody offer for experimental applications?

FITC-conjugated SHISA9 antibodies provide several methodological advantages:

• Direct visualization without secondary antibody requirements, reducing background and cross-reactivity issues.

• Compatibility with multi-labeling experiments when combined with antibodies conjugated to spectrally distinct fluorophores.

• Suitability for live-cell imaging applications when using non-perturbing antibodies against extracellular epitopes.

• Quantitative capabilities when calibrated with fluorescence standards.

• Time efficiency in protocols by eliminating secondary antibody incubation steps.

How can immunoprecipitation protocols be optimized specifically for SHISA9 in different brain regions?

Optimizing immunoprecipitation for SHISA9 requires consideration of region-specific protein expression levels and interaction dynamics:

Methodological approach for mouse brain tissue:

• Homogenize fresh tissue in 25 mM HEPES/NaOH (pH 7.4), 0.32 M sucrose with protease inhibitors using 12 up-and-down motions with a potter homogenizer at 900 rpm .

• Perform differential centrifugation: 1,000g for 10 minutes followed by 100,000g for 2 hours to obtain the membrane-enriched P2-fraction .

• Resuspend P2-fraction to 10 μg/μL protein and solubilize with equal volume of lysis buffer containing 2% DDM .

• After clearing insoluble material (20,000g, 15 min), incubate with anti-SHISA9 antibody (approximately 2-4 μg antibody per sample) overnight at 4°C .

• Add protein A/G beads, incubate for 1 hour, then wash 4 times with buffer containing 0.1% DDM .

Research demonstrates this protocol successfully identifies SHISA9-PSD95 interactions in both hippocampus and cortex, but adjustments in antibody concentration or detergent type may be necessary for regions with different expression levels or lipid composition .

What experimental controls validate the specificity of SHISA9 antibody signal?

Multiple complementary controls should be implemented to validate SHISA9 antibody specificity:

• Peptide competition assay: Pre-incubate antibody with excess immunizing peptide to block specific binding sites. The research describes a similar approach using biotinylated Shisa9 peptide and competition with TAT-tagged Shisa9 peptides that reduced specific interactions by approximately 50% .

• Genetic controls: Use tissue from SHISA9 knockout animals as negative controls.

• Antibody controls:

  • Isotype control antibodies

  • Multiple antibodies targeting different SHISA9 epitopes

  • Concentration gradients to establish signal-to-noise ratios

• Domain-specific validation: The research utilized Shisa9ΔEVTV constructs lacking the C-terminal PDZ-binding motif to demonstrate the specificity of interactions with PDZ domain-containing proteins .

• Cross-methodology validation: Compare results across multiple techniques (e.g., co-immunoprecipitation, proximity ligation, immunofluorescence) to confirm consistent localization and interaction patterns.

How do SHISA9 molecular interactions influence AMPA receptor function?

SHISA9 interactions with PDZ domain-containing proteins critically influence AMPA receptor function in several ways:

• AMPAR current kinetics: Research demonstrates that disrupting SHISA9 C-terminal interactions using mimetic peptides significantly accelerates AMPAR current decay time in hippocampal neurons (from 6.27±0.29 ms to 5.21±0.28 ms) .

• Recovery from desensitization: SHISA9 PDZ interactions regulate paired-pulse facilitation, with disruption reducing facilitation at 20-100ms intervals, indicating slower recovery from desensitization .

• Network activity modulation: Interference with SHISA9-PDZ interactions significantly increases the power of hippocampal network oscillations, demonstrating that these molecular interactions influence not only individual synapses but also coordinated network activity .

• Synaptic anchoring: Interactions with PSD95, PSD93, PICK1, GRIP1, and Lin7b likely anchor SHISA9 at the postsynaptic density, positioning it to modulate AMPA receptor properties .

The research demonstrates that these effects are specifically mediated through the C-terminal EVTV motif, as a TAT-Shisa9ΔEVTV peptide lacking this motif did not affect AMPAR properties .

What methodological approach can distinguish between effects of different SHISA9-interacting proteins?

Distinguishing between effects of different SHISA9 interactors requires multi-faceted experimental approaches:

• Domain-specific disruption: Design specific peptide mimetics or mutations targeting interaction interfaces between SHISA9 and individual binding partners. The research successfully used TAT-Shisa9WT peptide to broadly disrupt PDZ interactions , but more selective approaches include:

  • Point mutations in specific binding motifs

  • Interface-specific blocking antibodies

  • Selective knockdown of individual interactors

• Sequential co-immunoprecipitation:

  • First IP: Pull down SHISA9 with its antibody

  • Second IP: Re-immunoprecipitate with antibodies against specific interactors

• Proximity-dependent labeling:

  • Express SHISA9 fused to BioID or APEX2

  • Identify spatially restricted interaction partners through biotinylation

  • Compare results across different conditions or brain regions

• Functional correlation:

  • Combine electrophysiological recordings with molecular manipulations

  • Correlate changes in AMPAR properties with specific disruptions

  • The research demonstrated distinct effects on decay time and paired-pulse facilitation, suggesting multiple functional consequences of SHISA9 interactions

How does synaptic activity regulate SHISA9 interactions with its binding partners?

While the primary research didn't directly investigate activity-dependent regulation of SHISA9 interactions, several experimental approaches can address this question:

• Activity-dependent biochemical analysis:

  • Compare SHISA9 co-immunoprecipitation profiles before and after stimulation protocols

  • Use phospho-specific antibodies to detect post-translational modifications

  • Implement crosslinking before solubilization to capture transient interactions

• Live imaging approaches:

  • Express fluorescently-tagged SHISA9 and interacting partners

  • Monitor redistribution following synaptic stimulation

  • Use FRET sensors to detect conformational changes in protein complexes

• Functional correlations:

  • The research shows that disrupting SHISA9-PDZ interactions affects hippocampal network oscillations, suggesting activity-dependent roles

  • Design experiments examining SHISA9 effects during different activity states

  • Test whether SHISA9 interactions change during synaptic plasticity induction

• Comparative analysis across brain regions:

  • The research confirmed SHISA9-PSD95 interactions in both hippocampus and cortex

  • Compare interaction profiles across regions with different baseline activity

What tissue preparation protocols optimize SHISA9 antibody labeling in immunohistochemistry?

Optimal tissue preparation for SHISA9 immunohistochemistry with FITC-conjugated antibodies:

• Fixation protocol:

  • Perfusion with ice-cold PBS followed by 4% paraformaldehyde

  • Post-fixation for 2-4 hours (avoid overfixation which may mask epitopes)

  • Cryoprotection in 30% sucrose until tissue sinks

• Sectioning options:

  • Free-floating sections (40μm) for optimal antibody penetration

  • Thin sections (10-20μm) on slides for co-localization studies

  • Vibratome sections for acute preparations

• Antigen retrieval methods:

  • Heat-mediated: Citrate buffer (pH 6.0) at 80°C for 20 minutes

  • Enzymatic: Brief proteinase K treatment (optimize concentration)

  • Test multiple methods as PDZ domain epitopes may require specific approaches

• Permeabilization and blocking:

  • 0.3% Triton X-100 in PBS for membrane permeabilization

  • 10% normal serum (from species unrelated to antibody) for 2 hours

  • Include 0.1% glycine to block free aldehyde groups

• Antibody incubation parameters:

  • FITC-conjugated antibodies are light-sensitive; perform incubations in darkness

  • Extended incubation (24-48h at 4°C) may improve signal-to-noise ratio

  • Include 0.1% Triton X-100 and 2% normal serum in antibody diluent

What factors affect the quantitative analysis of SHISA9 expression using FITC-conjugated antibodies?

Quantitative analysis of SHISA9 expression using FITC-conjugated antibodies requires attention to multiple technical factors:

• Signal calibration:

  • Include calibration standards in each experiment

  • Use identical acquisition parameters across all samples

  • Implement background subtraction using no-primary controls

• FITC-specific considerations:

  • Photobleaching occurs more rapidly than with more stable fluorophores

  • Signal intensity decreases with exposure time

  • pH sensitivity affects fluorescence intensity (optimal at pH 7.4-8.0)

• Sample preparation variables:

  • Fixation duration affects epitope accessibility

  • Antibody concentration determines signal intensity (establish standard curve)

  • Section thickness influences signal penetration

• Imaging parameters:

  • Objective magnification and numerical aperture affect resolution

  • Detector gain settings influence signal-to-noise ratio

  • Z-stack sampling for volumetric quantification

• Analysis methodology:

  • Define consistent region of interest selection criteria

  • Establish threshold criteria for positive signal identification

  • Normalize to internal standards (e.g., neuronal markers or total protein)

How can peptide competition assays validate SHISA9 antibody specificity in different experimental contexts?

Peptide competition assays provide powerful validation of antibody specificity across multiple experimental contexts:

Implementation methodology:

• For immunohistochemistry/immunofluorescence:

  • Prepare two identical tissue sections

  • Pre-incubate FITC-conjugated SHISA9 antibody with 100-fold molar excess of immunizing peptide for 2 hours at room temperature

  • Apply blocked antibody to one section and unblocked antibody to the other

  • Process samples identically and compare signal patterns

• For biochemical applications:

  • The research demonstrated a similar approach using biotinylated Shisa9 peptide coupled to NeutrAvidin beads

  • Pre-incubate with competing peptides (TAT-Shisa9WT) to disrupt specific interactions

  • This approach reduced interaction by approximately 50%, confirming specificity

  • Include control peptides (the research used TAT-Shisa9ΔEVTV and TAT-scrambled) to verify specificity

• Quantitative assessment:

  • Calculate percent signal reduction with blocked versus unblocked antibody

  • Significant reduction (typically >80%) indicates specific binding

  • Partial reduction may indicate multiple epitopes or cross-reactivity

• Controls and variations:

  • Include unrelated peptide pre-incubation as negative control

  • Test concentration-dependent effects with peptide titration

  • Compare results between antibodies targeting different SHISA9 epitopes

What approaches can integrate SHISA9 antibody labeling with electrophysiological studies?

Integration of SHISA9 antibody labeling with electrophysiology requires specialized approaches:

• Sequential experimental design:

  • Perform electrophysiological recordings in acute brain slices

  • Include biocytin in recording pipette to mark recorded neurons

  • Post-recording fixation and immunolabeling with FITC-conjugated SHISA9 antibody

  • This approach allows direct correlation between SHISA9 expression and functional properties

• Mimetic peptide approach:

  • The research successfully used TAT-Shisa9WT peptide to disrupt SHISA9 interactions during electrophysiological recordings

  • TAT-fusion peptides penetrate neurons in acute slices

  • Record AMPAR currents before and after peptide application

  • Include control peptides (TAT-Shisa9ΔEVTV) as demonstrated in the research

• Technical parameters affecting integration:

  • Peptide concentration: The research used 10μM TAT-Shisa9 peptides for electrophysiology

  • Incubation time: Allow 10-20 minutes for peptide penetration

  • Recording configuration: The research used whole-cell recordings from dentate gyrus granule cells

  • Stimulation paradigm: The research stimulated lateral perforant path inputs

• Measurable parameters:

  • AMPAR current decay time: Disrupting SHISA9 interactions accelerated decay (from 6.27±0.29 ms to 5.21±0.28 ms)

  • Paired-pulse facilitation: Reduced after SHISA9 interaction disruption (from 1.62±0.04 to 1.27±0.02 at 50ms interval)

  • Network oscillations: Power increased after SHISA9 interaction disruption

What protein-protein interaction assays can validate SHISA9 binding partners identified in initial screening?

Multiple complementary protein-protein interaction assays can validate SHISA9 interactors:

• Co-immunoprecipitation approaches:

  • Forward IP: The research immunoprecipitated Shisa9 and detected PSD95

  • Reverse IP: Immunoprecipitate candidate interactors and probe for SHISA9

  • Expression system validation: The research validated interactions by co-expressing HA-tagged Shisa9WT or Shisa9ΔEVTV with V5-tagged interactors in HEK293T cells

• Pull-down assays:

  • The research used biotinylated Shisa9 peptides coupled to NeutrAvidin beads to pull down recombinant PSD95

  • This approach confirmed direct interaction without intermediate proteins

  • Competition with TAT-Shisa9WT peptide reduced binding by approximately 50%

• Domain mapping strategies:

  • The research created Shisa9ΔEVTV construct lacking the PDZ-binding motif

  • This construct failed to interact with PDZ domain-containing proteins

  • Similar approaches can map other interaction interfaces

• Proximity-based assays:

  • Proximity ligation assay (PLA): Detects protein interactions within 40nm in fixed cells/tissues

  • FRET/BRET: Measures direct interactions between fluorescently tagged proteins

  • BiFC: Visualizes protein interactions through complementation of split fluorescent proteins

How should FITC-conjugated antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of FITC-conjugated SHISA9 antibodies preserves their performance:

• Storage conditions:

  • Temperature: -20°C for long-term; 4°C for working aliquots (up to 2 weeks)

  • Aliquoting: Prepare single-use aliquots to avoid freeze-thaw cycles (limit to <5)

  • Protection from light: Store in amber tubes or wrap in aluminum foil

  • Buffer composition: PBS (pH 7.4) with 0.05-0.1% sodium azide and 0.1% BSA

• Handling guidelines:

  • Light exposure: Minimize exposure to light during all handling steps

  • Temperature equilibration: Allow to reach room temperature before opening

  • Centrifugation: Brief spin before opening to collect liquid

  • Contamination prevention: Use clean pipette tips and tubes

• Working dilution preparation:

  • Freshness: Prepare fresh dilutions for each experiment

  • Diluent: Use filtered buffers with carrier protein (0.1-0.5% BSA)

  • Filtration: Consider 0.22μm filtration if any particulates are present

  • Storage: Keep diluted antibody at 4°C and use within 24 hours

• FITC-specific considerations:

  • Photobleaching sensitivity: FITC bleaches more rapidly than many other fluorophores

  • pH sensitivity: Optimal fluorescence at pH 7.4-8.0

  • Quenching agents: Avoid reducing agents in buffers

  • Quality control: Test each lot on standard samples before experiments

What factors influence the specificity of SHISA9 detection in complex neural tissue samples?

Several factors influence SHISA9 detection specificity in neural tissues:

• Antibody characteristics:

  • Epitope location: The PDZ-binding C-terminus is unique to SHISA9 and provides specificity

  • Clonal source: Monoclonal antibodies often provide higher specificity

  • Validation: Antibodies should be validated against SHISA9 knockout tissue

• Tissue preparation factors:

  • Fixation parameters: Overfixation can mask epitopes while insufficient fixation reduces structural integrity

  • Autofluorescence: Brain tissue contains lipofuscin that autofluoresces in the same range as FITC

  • Antigen retrieval: PDZ domain epitopes may require specific retrieval methods

• Protocol optimization:

  • Blocking stringency: Include 10% normal serum and 0.1-0.3% Triton X-100

  • Antibody concentration: Titrate to determine optimal signal-to-noise ratio

  • Incubation time: Extended incubation (24-48h at 4°C) may improve specificity

• Controls for specificity:

  • Peptide competition: Pre-incubate with immunizing peptide as demonstrated in the research

  • Genetic controls: SHISA9 knockout tissue provides definitive negative control

  • Signal correlation: Compare with mRNA expression patterns

• Detection system considerations:

  • Direct conjugation: FITC-conjugated primary antibodies eliminate secondary antibody cross-reactivity

  • Signal amplification: Must balance with potential increase in background

  • Multiple labeling: Consider spectral overlap when combining with other fluorophores

How do experimental conditions affect SHISA9-PDZ domain interactions in functional studies?

Experimental conditions significantly influence SHISA9-PDZ domain interactions:

• Temperature effects:

  • The research conducted protein interaction studies at room temperature

  • Electrophysiological recordings were performed at room temperature

  • Temperature affects binding kinetics and may influence interaction stability

• Buffer composition factors:

  • Ionic strength: Higher salt concentrations can disrupt electrostatic interactions

  • Divalent cations: Ca²⁺ and Mg²⁺ concentrations affect protein conformation

  • pH: PDZ domain interactions typically have pH-dependent stability

  • Detergents: The research used DDM (dodecyl maltoside) to solubilize membrane proteins while preserving interactions

• Peptide parameters:

  • Concentration: The research used 10μM TAT-Shisa9 peptides for functional studies

  • Penetration time: Allow sufficient incubation (10-20 minutes) for peptide uptake

  • Specificity controls: The research used TAT-Shisa9ΔEVTV and TAT-scrambled peptides

• Activity-dependent considerations:

  • The research demonstrated effects on hippocampal network oscillations, suggesting activity-dependent functions

  • Baseline activity: Different recording conditions may affect SHISA9 interaction status

  • Stimulation paradigms: The research examined lateral perforant path stimulation

  • Neuronal subtype: The research focused on dentate gyrus granule cells

• Readout selection:

  • Direct biochemical measurement: Pull-down assays as used in the research

  • Functional correlates: AMPAR current decay time and paired-pulse facilitation

  • Network effects: Power of hippocampal oscillations