shisa9b Antibody

What is Shisa9 and why are antibodies against it important for neuroscience research?

Shisa9 (initially named CKAMP44) is a type-I transmembrane protein that functions as an auxiliary subunit of AMPA-type glutamate receptors. It contains a C-terminal PDZ domain that interacts with cytosolic proteins and modulates the physiological properties of AMPA receptors . Antibodies against Shisa9 are critical research tools that enable detection, localization, and functional analysis of this protein in neuronal tissues. These antibodies help researchers investigate how Shisa9 influences synaptic transmission, as studies have shown that Shisa9's C-terminal interactions affect AMPA receptor-mediated synaptic currents and hippocampal network activity .

How do Shisa9 antibodies differ from other neuronal protein antibodies in terms of specificity and cross-reactivity?

Shisa9 antibodies are designed to recognize specific epitopes on the Shisa9 protein, which has unique structural and functional properties compared to other neuronal proteins. Like other highly specific antibodies, Shisa9 antibodies should undergo rigorous validation to ensure they do not cross-react with other members of the Shisa protein family or unrelated proteins. When developing or selecting Shisa9 antibodies, researchers should consider epitope mapping, validation across multiple experimental platforms (Western blot, immunoprecipitation, immunohistochemistry), and testing in both wild-type and knockout tissues to confirm specificity. Unlike some widely characterized antibodies like those targeting PSD95, Shisa9 antibodies may require more extensive validation due to the relatively recent characterization of this protein in the scientific literature .

What are the recommended specimen collection and storage conditions for optimal Shisa9 antibody performance?

For optimal Shisa9 antibody performance, researchers should follow specimen collection and handling procedures similar to those used for other neuronal proteins. Based on standard protocols for antibody-based assays, tissue samples should be collected fresh and processed quickly to preserve protein integrity. For brain tissue samples, rapid dissection and immediate flash-freezing in liquid nitrogen or fixation (depending on the intended application) is recommended. For storage conditions, tissue samples should be kept at -80°C for long-term storage if unfixed, while fixed samples can be stored according to standard immunohistochemistry protocols. When preparing lysates for immunoprecipitation or Western blot analysis, as demonstrated in the Shisa9 study, appropriate lysis buffers containing protease inhibitors should be used to prevent protein degradation .

What are the optimal conditions for using Shisa9 antibodies in co-immunoprecipitation experiments to study protein interactions?

For co-immunoprecipitation experiments studying Shisa9 interactions with other proteins, researchers should consider the following methodological approach based on published work: First, prepare tissue lysates from regions known to express Shisa9, such as the hippocampus and cortex, using detergent-based lysis buffers that preserve protein-protein interactions. Add anti-Shisa9 antibody to the lysates at an experimentally determined optimal concentration (typically 2-5 μg of antibody per mg of total protein) . Include appropriate controls, such as IgG from the same species as the Shisa9 antibody. Allow binding to occur (typically 1-2 hours at 4°C with gentle rotation), then add protein A/G beads to capture the antibody-protein complexes. After thorough washing to remove non-specific binding, elute the complexes and analyze by Western blotting using antibodies against potential interacting partners. This approach has successfully demonstrated that Shisa9 forms complexes with PSD95 in the hippocampus and cortex .

How should researchers optimize immunohistochemistry protocols for Shisa9 detection in different brain regions?

Optimizing immunohistochemistry protocols for Shisa9 detection requires careful consideration of fixation methods, antigen retrieval, antibody concentration, and detection systems. For brain tissue sections, paraformaldehyde fixation (4%) is typically suitable, but optimization may be necessary depending on the specific epitope recognized by the Shisa9 antibody. Researchers should test different antigen retrieval methods (heat-induced in citrate buffer pH 6.0 or Tris-EDTA pH 9.0) to enhance antibody binding while preserving tissue morphology. The Shisa9 antibody concentration should be titrated (typically starting with 1:100 to 1:1000 dilutions) to determine optimal signal-to-noise ratio. Since Shisa9 is expressed in specific brain regions including the hippocampus and cortex , include these regions as positive controls and regions with low/no expression as negative controls. When possible, validate staining patterns by comparing with RNA expression data or using tissue from Shisa9 knockout animals as specificity controls.

What approaches are recommended for quantitative analysis of Shisa9 expression levels in comparative studies?

For quantitative analysis of Shisa9 expression levels, researchers should employ multiple complementary techniques to ensure robust results. Western blotting with carefully validated Shisa9 antibodies provides a reliable method for comparing protein levels across different experimental conditions or brain regions. For accurate quantification, researchers should: 1) Ensure equal protein loading using total protein normalization rather than single housekeeping proteins; 2) Include a standard curve of recombinant Shisa9 protein when absolute quantification is needed; 3) Use fluorescence-based detection systems that offer broader linear range than chemiluminescence; and 4) Perform biological replicates (n≥3) with appropriate statistical analysis. For spatial expression analysis, quantitative immunohistochemistry with standardized image acquisition parameters and analysis algorithms can provide region-specific expression data. Alternative approaches include quantitative PCR for mRNA expression analysis or mass spectrometry-based proteomics for unbiased protein quantification, which can complement antibody-based methods .

How can researchers distinguish between specific and non-specific binding when using Shisa9 antibodies in Western blot applications?

To distinguish between specific and non-specific binding in Western blot applications using Shisa9 antibodies, researchers should implement several validation strategies. First, confirm the expected molecular weight of Shisa9 (approximately 44 kDa as initially named CKAMP44) while recognizing that post-translational modifications may alter migration patterns. Include positive controls (tissues known to express Shisa9, such as hippocampus and cortex) and negative controls (tissues with minimal Shisa9 expression or samples from Shisa9 knockout animals when available). Perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide, which should eliminate specific bands. Consider testing multiple antibodies targeting different epitopes of Shisa9, as consistent detection across antibodies increases confidence in specificity. When non-specific bands appear, optimize blocking conditions, antibody concentrations, and washing stringency. Additionally, comparing the pattern of protein detection with known mRNA expression patterns across tissues can provide further validation of antibody specificity.

What are the most common technical challenges when using Shisa9 antibodies in electrophysiology studies, and how can they be addressed?

When using Shisa9 antibodies in electrophysiology studies, researchers may encounter several technical challenges. First, ensuring antibody access to the target protein in intact tissue preparations can be difficult. Researchers should optimize tissue preparation methods, including slice thickness and incubation times with antibodies. Second, maintaining neuronal viability during antibody application is crucial; use physiological solutions and minimize exposure times. Third, distinguishing antibody-specific effects from non-specific effects requires rigorous controls, including using non-immune IgG and Fab fragments of the Shisa9 antibody to rule out effects due to antibody size or Fc receptor interactions. Fourth, when using antibodies to disrupt protein interactions, as done with Shisa9 C-terminal mimetic peptides , researchers should validate the specificity of this approach using multiple concentrations and scrambled peptide controls. The successful application of Shisa9 C-terminal mimetic peptides in studying AMPA receptor-mediated synaptic currents demonstrates the potential of this approach when properly controlled .

How should researchers address inconsistent or contradictory results when using different lots of Shisa9 antibodies?

Inconsistent or contradictory results between different lots of Shisa9 antibodies can significantly impact research findings. To address this issue, researchers should implement a systematic approach to antibody validation and experimental consistency. First, maintain detailed records of antibody lot numbers, validation data, and experimental conditions for each lot used. When switching to a new lot, perform side-by-side comparisons with the previous lot across multiple applications (Western blot, immunoprecipitation, immunohistochemistry) using identical samples and protocols. If discrepancies are observed, conduct additional validation experiments, including peptide competition assays and testing in knockout tissue when available. Consider using pooled samples as internal controls that can be run across multiple experiments to normalize for lot-to-lot variations. For critical experiments, pre-order and reserve sufficient quantities of a single, well-validated lot. Additionally, contact the antibody manufacturer for technical support and lot-specific validation data, as manufacturing processes may change over time. When reporting results, clearly document the specific antibody lot used and include validation data in supplementary materials to enhance reproducibility.

How can Shisa9 antibodies be utilized to investigate the protein's role in synaptic plasticity mechanisms?

Shisa9 antibodies offer powerful tools for investigating this protein's role in synaptic plasticity through multiple advanced approaches. Researchers can employ function-blocking antibodies or Fab fragments that specifically target the extracellular domain of Shisa9 to acutely disrupt its interactions with AMPA receptors during electrophysiological recordings of synaptic plasticity. This approach has revealed that disrupting C-terminal scaffolding interactions of Shisa9 using mimetic peptides results in faster decay time of glutamatergic AMPA receptor-mediated synaptic currents and reduced paired-pulse facilitation in the lateral perforant path of the mouse hippocampus . For in vivo studies, researchers can combine viral-mediated expression of tagged Shisa9 constructs with anti-tag antibodies to visualize activity-dependent changes in Shisa9 localization during plasticity induction. Time-resolved immunoprecipitation with phospho-specific antibodies can reveal post-translational modifications of Shisa9 following synaptic activity. Super-resolution microscopy with Shisa9 antibodies, combined with markers for AMPA receptors and scaffold proteins, can provide nanoscale information about dynamic reorganization of these complexes during plasticity events, building on the known interactions between Shisa9 and PDZ domain-containing scaffold proteins like PSD95 .

What are the methodological considerations for using Shisa9 antibodies in proximity ligation assays to study protein-protein interactions in situ?

Proximity ligation assay (PLA) offers a powerful method to detect and localize Shisa9 interactions with binding partners directly in tissue sections or cultured neurons. When designing PLA experiments with Shisa9 antibodies, researchers should consider several methodological factors. First, antibody selection is critical—use antibodies raised in different species against Shisa9 and potential binding partners (such as the identified PDZ domain-containing interactors: PSD93, PSD95, MPP5, GRIP1, PICK1, Lin7b, and GIPC1) . Validate antibody specificity individually before attempting PLA. Second, tissue preparation must preserve both protein epitopes and spatial relationships; optimize fixation conditions that maintain membrane protein structure while allowing antibody access. Third, include appropriate controls: positive controls using known interacting proteins, negative controls omitting one primary antibody, and biological controls in tissues where the interaction is absent or disrupted (such as using tissues expressing Shisa9 with mutated PDZ binding motifs). Fourth, for quantitative analysis, establish objective criteria for identifying and counting PLA signals, and relate these to appropriate reference markers (such as synaptic markers). Fifth, consider the spatial resolution limitations of PLA (~40 nm) when interpreting results, particularly in densely packed structures like the postsynaptic density where Shisa9 and its interactors are localized .

How can researchers effectively use Shisa9 antibodies in combination with other methodologies to study its role in neurological disorders?

Integrating Shisa9 antibodies with complementary methodologies creates powerful approaches for investigating its role in neurological disorders. Researchers can combine immunohistochemistry using validated Shisa9 antibodies with electrophysiological recordings in brain slices from disease models to correlate expression patterns with functional alterations in AMPA receptor-mediated transmission. This approach builds on findings that Shisa9's C-terminal interactions affect synaptic function and hippocampal network activity . For human studies, researchers can develop protocols for Shisa9 detection in post-mortem brain tissue or cerebrospinal fluid, potentially revealing disease-specific alterations. In animal models, viral-mediated expression of dominant-negative Shisa9 constructs combined with behavioral testing can establish causal relationships between Shisa9 dysfunction and disease-relevant phenotypes. Single-cell transcriptomics paired with Shisa9 immunolabeling can identify cell populations with altered Shisa9 expression in disease states. Additionally, researchers can develop phospho-specific antibodies against Shisa9 to track disease-related changes in its post-translational modifications. When applying these approaches to neurological disorders, researchers should consider disease-specific factors such as altered protein degradation, inflammation, or blood-brain barrier dysfunction that might affect antibody performance or data interpretation.

How can researchers adapt Shisa9 antibodies for use in high-throughput screening applications?

Adapting Shisa9 antibodies for high-throughput screening requires optimization of antibody performance across automated platforms while maintaining specificity and sensitivity. Researchers should first evaluate multiple Shisa9 antibody clones to identify those with optimal performance characteristics including low background, high signal-to-noise ratio, and batch-to-batch consistency. Conjugate selected antibodies directly with fluorophores, biotin, or HRP to eliminate secondary antibody steps and improve workflow efficiency. For cell-based high-throughput screens, optimize fixation, permeabilization, and staining protocols in 96- or 384-well formats, focusing on minimizing volumes and incubation times while maintaining signal quality. Consider developing homogeneous assay formats (no-wash steps) using technologies like TR-FRET (time-resolved fluorescence resonance energy transfer) with labeled Shisa9 antibodies. For protein-protein interaction screens, adapt Shisa9 antibodies for use in automated co-immunoprecipitation workflows or bead-based multiplexed assays to simultaneously detect multiple binding partners identified in yeast two-hybrid screens . Implement quality control measures including Z'-factor determination and coefficient of variation analysis across plates. When screening compound libraries for modulators of Shisa9 function or expression, include positive controls (known PDZ domain inhibitors) and negative controls to ensure assay robustness and reproducibility.

What considerations are important when developing phospho-specific antibodies against Shisa9 for signaling pathway studies?

Developing phospho-specific antibodies against Shisa9 for signaling studies requires careful attention to several key factors. First, researchers must identify physiologically relevant phosphorylation sites through phosphoproteomic analysis of neuronal tissues or cells expressing Shisa9, focusing particularly on activity-dependent modifications that might regulate its interactions with PDZ domain-containing proteins . Once candidate sites are identified, design phosphopeptide immunogens that include 10-15 amino acids surrounding the phosphorylation site, with the phosphorylated residue centrally positioned. For each phospho-site, generate paired antibodies—one phospho-specific and one that recognizes the same region regardless of phosphorylation status—to enable calculation of phosphorylation stoichiometry. During antibody production and purification, implement rigorous validation protocols including peptide competition assays with phosphorylated and non-phosphorylated peptides, testing in samples treated with phosphatases, and evaluation in tissues from kinase knockout models or following pharmacological kinase inhibition. For signaling pathway studies, optimize detection methods for low-abundance phosphorylated species, potentially incorporating signal amplification strategies or highly sensitive detection systems. Consider temporal dynamics of phosphorylation events, which may require time-course experiments following specific stimuli relevant to Shisa9 function in AMPA receptor modulation .

How can novel imaging approaches enhance the utility of Shisa9 antibodies in studying receptor trafficking and synaptic localization?

Novel imaging approaches significantly expand the utility of Shisa9 antibodies for studying receptor trafficking and synaptic localization with unprecedented resolution and molecular specificity. Super-resolution microscopy techniques (STORM, PALM, STED) combined with Shisa9 antibodies can resolve the nanoscale organization of Shisa9 relative to AMPA receptors and scaffolding proteins like PSD95 within the postsynaptic density, revealing how these spatial relationships correlate with synaptic strength. For live imaging applications, researchers can develop recombinant single-chain antibody fragments (scFvs) against Shisa9 that function intracellularly as "intrabodies" when fused to fluorescent proteins, allowing real-time visualization of endogenous Shisa9 trafficking in living neurons without fixation artifacts. Expansion microscopy, which physically enlarges specimens, can be combined with conventional Shisa9 antibodies to achieve super-resolution-like images on standard microscopes, particularly valuable for mapping the complex protein interaction network of Shisa9 in intact circuits. Multi-epitope imaging using antibodies against different domains of Shisa9 can reveal conformational changes associated with protein-protein interactions or receptor modulation. Correlative light and electron microscopy (CLEM) using Shisa9 antibodies conjugated to both fluorophores and electron-dense particles allows researchers to connect functional imaging data with ultrastructural context. These advanced imaging approaches will provide critical insights into how Shisa9's interactions with AMPA receptors and PDZ domain-containing proteins regulate receptor trafficking and synaptic function.