SHISA9 Antibody, Biotin conjugated

What is SHISA9 and what functional roles does it play in neuronal systems?

SHISA9 (also known as CKAMP44) functions as an auxiliary subunit of AMPA-type glutamate receptors that modulates their physiological properties. It regulates short-term neuronal synaptic plasticity in the dentate gyrus and associates with AMPA receptors in synaptic spines where it promotes receptor desensitization at excitatory synapses . This type-I transmembrane protein contains a C-terminal PDZ domain that interacts with cytosolic scaffolding proteins .

SHISA9 demonstrates brain-specific expression patterns, being predominantly found in neurons across most brain structures during both embryonic and postnatal development . It serves as a component of AMPA receptor complexes that include the AMPA receptor itself, CACNG2, SHISA9, and lower levels of DLG7 . Its cellular distribution includes cell projections, synaptic junctions, and insertion as a single-pass type I membrane protein .

What are the distinguishing characteristics of biotin-conjugated SHISA9 antibodies?

Biotin-conjugated SHISA9 antibodies offer several methodological advantages over unconjugated alternatives:

The biotin conjugation enables high-affinity binding with streptavidin or avidin molecules, enhancing detection sensitivity in immunoassays while eliminating the need for species-specific secondary antibodies .

How does SHISA9 contribute to AMPA receptor function at the molecular level?

SHISA9 modulates AMPA receptor function through several mechanisms:

• It promotes AMPA receptor desensitization at excitatory synapses, affecting the kinetics of receptor responses

• Through its C-terminal PDZ domain interactions, it influences glutamatergic AMPA receptor-mediated synaptic currents, specifically affecting decay time parameters

• Disruption of these PDZ interactions between SHISA9 and its binding partners affects paired-pulse facilitation and hippocampal network activity

• It functions within a protein complex that includes AMPA receptors, CACNG2, and varying levels of DLG7

What protocol optimizations are recommended for co-immunoprecipitation of SHISA9-containing complexes?

For effective co-immunoprecipitation of SHISA9 complexes from brain tissue, researchers should implement the following methodological approach:

• Homogenize mouse cortex or hippocampus with a potter and piston at 900 rpm on ice, with twelve up-and-down motions in homogenization buffer (25 mM HEPES/NaOH pH 7.4, 0.32 M sucrose, protease inhibitor)

• Perform sequential centrifugation: first at 1,000 g for 10 minutes at 4°C, then centrifuge the supernatant at 100,000 g for 2 hours to obtain the P2-fraction

• Resuspend the pellet in HEPES buffer to 10 μg/μL protein concentration and mix with an equal volume of lysis buffer containing 2% n-dodecyl β-d-maltoside (DDM)

• After incubation (45 minutes rotation) at 4°C, centrifuge at 20,000 g for 15 minutes

• For immunoprecipitation, add anti-Shisa9 antibody (approximately 12 μg) to the supernatant and incubate overnight with rotation at 4°C

• Add agarose-protein A/G beads and incubate for 1 hour at 4°C, followed by washing four times in lysis buffer with 0.1% DDM

• Elute proteins with SDS sample buffer for subsequent analysis

This protocol accounts for the challenging nature of solubilizing postsynaptic density proteins while maintaining protein complex integrity .

How can researchers validate the specificity of interactions between SHISA9 and PDZ domain-containing proteins?

To validate SHISA9 interactions with PDZ domain-containing proteins, researchers should employ multiple complementary approaches:

• Yeast two-hybrid screening: Use the Shisa9 cytoplasmic domain as bait to identify potential interacting partners, followed by direct two-hybrid assays with representative clones to confirm specificity

• PDZ-ligand motif mutation studies: Test interactions after deletion of the C-terminal PDZ-ligand motif (EVTV) to verify the interaction mechanism. Research demonstrates that removal of this sequence completely disrupts cell growth for PDZ domain-containing proteins in yeast two-hybrid systems

• Co-immunoprecipitation validation: Express HA-tagged Shisa9WT or HA-Shisa9ΔEVTV proteins with V5-tagged interactors in HEK293T cells, then perform co-immunoprecipitation with anti-HA antibody to confirm PDZ domain-dependent interactions

• Peptide competition assays: Utilize biotin-tagged Shisa9 C-terminal peptides (e.g., biotin-HFPPTQPYFITNSKTEVTV) and TAT-tagged Shisa9 peptides to disrupt protein-protein interactions and confirm binding specificity

• Endogenous complex verification: Immunoprecipitate native Shisa9 complexes from brain tissue followed by immunoblotting for suspected interaction partners, such as PSD95

What Western blotting parameters should be optimized when using biotin-conjugated SHISA9 antibodies?

For optimal Western blotting results with biotin-conjugated SHISA9 antibody:

• Transfer proteins overnight at 40V onto PVDF membrane for optimal transfer of higher molecular weight proteins

• Block membranes with 5% milk in TBST to reduce non-specific binding

• Use the recommended 1:10,000 dilution of the biotin-conjugated antibody

• Incubate membrane with primary antibody overnight at 4°C on a shaking platform

• Detect using streptavidin-conjugated enzymes (HRP or AP)

• Visualize using enhanced chemifluorescence or enhanced chemiluminescence femto according to manufacturer's instructions

• For protein bands of interest, verify specificity through comparison with appropriate molecular weight markers and control samples

How can researchers investigate the role of SHISA9 PDZ domain interactions in synaptic plasticity?

To investigate SHISA9's role in synaptic plasticity through its PDZ domain interactions:

• Peptide competition experiments: Use Shisa9 C-terminal mimetic peptides to disrupt scaffolding interactions and measure effects on glutamatergic AMPA receptor-mediated synaptic currents. Research indicates that in the absence of scaffolding interactions, synaptic currents in the lateral perforant path of mouse hippocampus exhibit faster decay time and reduced paired-pulse facilitation

• Electrophysiological recordings: Combine with molecular interventions (mimetic peptides, mutant constructs) to correlate SHISA9 PDZ domain interactions with functional changes in synaptic transmission

• Network activity analysis: Assess how disruption of PDZ interactions between SHISA9 and binding partners affects hippocampal network activity using multielectrode arrays or calcium imaging

• Protein complex analysis: Characterize the composition of SHISA9-containing AMPA receptor complexes under different plasticity conditions using co-immunoprecipitation followed by Western blotting or mass spectrometry

• Subcellular localization studies: Track changes in SHISA9 distribution relative to its binding partners during synaptic plasticity induction using immunocytochemistry or biochemical fractionation

What approaches can distinguish between distinct SHISA9 isoform functions?

To differentiate between SHISA9 isoform functions:

• Isoform-specific antibodies: Develop antibodies targeting unique epitopes in different isoforms (such as isoform X2), validated through Western blotting against recombinant isoforms

• Overexpression studies: Express individual SHISA9 isoforms in heterologous systems or neurons and compare their effects on AMPA receptor function using electrophysiology or biochemical analyses

• Domain swap experiments: Create chimeric constructs by swapping domains between isoforms to identify regions responsible for functional differences

• Isoform-specific knockdown: Design RNA interference strategies targeting unique sequences of specific isoforms to assess their differential contributions to synaptic function

• Proteomics approach: Use immunoprecipitation with isoform-specific antibodies coupled with mass spectrometry to identify differential protein interactions between isoforms

How can transcriptional and post-translational modifications of SHISA9 be investigated?

For comprehensive analysis of SHISA9 regulation:

• Phosphorylation studies: Use phospho-specific antibodies or mass spectrometry following immunoprecipitation to identify and characterize phosphorylation sites that may regulate SHISA9 function

• Developmental expression profiling: Apply the biotin-conjugated SHISA9 antibody in Western blotting of brain samples from different developmental stages to track expression changes

• Activity-dependent regulation: Examine changes in SHISA9 expression or modification following paradigms that induce synaptic plasticity or seizure activity

• Transcriptional analysis: Perform chromatin immunoprecipitation to identify transcription factors regulating SHISA9 expression during development and in response to neuronal activity

• Protein turnover assessment: Use pulse-chase experiments with metabolic labeling to determine SHISA9 half-life and how it might be regulated by neuronal activity or developmental stage

How should researchers address contradictory findings when studying SHISA9 interactions?

When faced with contradictory findings regarding SHISA9 interactions:

• Methodology considerations: Different experimental approaches have distinct limitations - yeast two-hybrid systems may identify more transient interactions than co-immunoprecipitation. For example, MPP5, GIPC1, and Dynlt3 were identified in yeast two-hybrid screens but not confirmed in co-immunoprecipitation experiments

• Solubilization challenges: The postsynaptic density is notoriously difficult to solubilize while maintaining protein complex integrity. Consider alternative detergents or solubilization conditions when working with endogenous complexes

• Interaction strength assessment: Use quantitative approaches such as surface plasmon resonance or microscale thermophoresis to measure binding affinities between SHISA9 and putative interactors

• Context-dependency: Evaluate whether interactions depend on specific post-translational modifications, cellular compartments, or activity states

• Biological versus technical variability: Distinguish between genuine biological heterogeneity and technical artifacts through adequate replications and controls

What strategies can overcome technical limitations in detecting low-abundance SHISA9 complexes?

To enhance detection of low-abundance SHISA9 complexes:

• Tissue enrichment: Focus on brain regions with highest SHISA9 expression, such as dentate gyrus of the hippocampus

• Subcellular fractionation: Isolate postsynaptic density fractions to enrich for SHISA9-containing complexes prior to analysis

• Signal amplification: Utilize biotin-streptavidin systems with tyramide signal amplification for immunohistochemistry applications

• Proximity ligation assays: Apply in situ proximity ligation to detect protein-protein interactions in fixed tissue with single-molecule sensitivity

• Mass spectrometry optimization: Implement targeted mass spectrometry approaches (such as selected reaction monitoring) to detect specific SHISA9 peptides with enhanced sensitivity

How should researchers interpret SHISA9 expression patterns across different experimental models?

When analyzing SHISA9 expression across experimental models:

• Species differences: Consider evolutionary conservation and potential functional divergence when comparing SHISA9 expression and function between species - the antibody shows reactivity with human, monkey, and mouse samples

• Developmental timing: Account for temporal expression patterns, as SHISA9 is expressed in most brain structures during both embryonic and postnatal development

• Cell-type specificity: Distinguish between neuronal subtypes, as expression may vary between excitatory and inhibitory neurons or across different brain regions

• Disease model considerations: In pathological conditions, altered SHISA9 expression may reflect compensatory mechanisms rather than causative factors

• Antibody validation: Cross-validate findings using multiple antibodies targeting different epitopes or complementary approaches such as in situ hybridization

How can SHISA9 antibodies be utilized to investigate AMPA receptor trafficking dynamics?

For studying AMPA receptor trafficking in relation to SHISA9:

• Live-cell imaging: Use fluorescently tagged SHISA9 constructs in combination with tagged AMPA receptor subunits to track co-trafficking in real-time

• Surface biotinylation assays: Measure surface expression of AMPA receptors in the presence or absence of SHISA9 overexpression or knockdown

• Antibody feeding experiments: Apply antibodies against extracellular epitopes of SHISA9 to track internalization kinetics in response to synaptic activity

• FRAP analysis: Perform fluorescence recovery after photobleaching to assess how SHISA9 affects the mobility of AMPA receptors within the membrane

• Super-resolution microscopy: Combine with immunolabeling using biotin-conjugated SHISA9 antibodies to visualize nanoscale organization of receptor complexes at synapses

What approaches can elucidate the contribution of SHISA9 to network-level neuronal activity?

To investigate SHISA9's contribution to network-level activity:

• Electrophysiological recordings: Apply peptide competition approaches with Shisa9 C-terminal mimetic peptides to disrupt PDZ interactions and measure effects on network activity. Research shows that disruption of the PDZ interactions between SHISA9 and its binding partners affects hippocampal network activity

• Circuit-specific manipulations: Use cell-type-specific genetic approaches to modulate SHISA9 expression in defined neuronal populations

• In vivo calcium imaging: Monitor network activity in brain regions with high SHISA9 expression during learning and memory tasks

• Computational modeling: Develop models incorporating SHISA9's effects on AMPA receptor kinetics to predict network-level consequences

• Behavioral correlates: Correlate changes in SHISA9 expression or function with behavioral outcomes in learning and memory paradigms

How might SHISA9 research contribute to understanding neuropsychiatric disorders?

SHISA9 research may advance understanding of neuropsychiatric disorders through:

• Expression analysis: Compare SHISA9 levels in postmortem brain samples from patients with conditions involving glutamatergic dysfunction (schizophrenia, autism, epilepsy) versus controls

• Genetic association studies: Investigate whether SHISA9 genetic variants correlate with neuropsychiatric conditions or treatment responses

• Animal models: Assess how SHISA9 manipulation affects behaviors relevant to psychiatric conditions (e.g., cognitive flexibility, social interaction, anxiety-like behaviors)

• Pharmacological interactions: Examine how drugs targeting glutamatergic transmission interact with SHISA9-mediated modulation of AMPA receptors

• Therapeutic potential: Explore whether enhancing or inhibiting SHISA9 function could represent a novel approach for modulating glutamatergic transmission in psychiatric disorders

The interacting partners of SHISA9 identified through yeast two-hybrid screening and validated by co-immunoprecipitation provide valuable targets for understanding its function in neuronal contexts:

These findings highlight the central role of SHISA9 in organizing multi-protein complexes at excitatory synapses through PDZ domain-mediated interactions.