SHISA9 Antibody, HRP conjugated

What is SHISA9 and why is HRP conjugation important for studying it?

SHISA9 (initially named CKAMP44) is an auxiliary subunit of AMPA-type glutamate receptors that modulates their physiological properties. It is a type-I transmembrane protein containing a C-terminal PDZ domain that interacts with various cytosolic proteins in the postsynaptic density . HRP (Horseradish Peroxidase) conjugation to SHISA9 antibodies enhances detection sensitivity in experimental procedures by providing an enzymatic amplification system for signal generation. This conjugation is particularly valuable for studying SHISA9 because the protein exists in relatively low abundance in specific brain regions, making high-sensitivity detection methods necessary . The conjugation process typically targets an average of 2-4 HRP molecules per antibody to maintain optimal detection capabilities while preserving antibody binding affinity .

What approaches are most effective for validating SHISA9 antibody specificity?

Validating SHISA9 antibody specificity requires multiple complementary approaches. First, researchers should perform western blotting against tissue from both wild-type and SHISA9 knockout models if available, looking for the absence of signal in knockout samples. Second, immunoprecipitation followed by mass spectrometry can confirm that the antibody captures the intended target with high specificity . Third, heterologous expression systems like HEK293T cells can be used by transfecting SHISA9 constructs and confirming antibody recognition . For brain tissue applications, immunohistochemistry patterns should be compared against known SHISA9 distribution, particularly in the hippocampus and cortex where SHISA9 expression has been well characterized . Cross-reactivity testing against related Shisa family proteins is essential to ensure specificity within this protein family.

What are the key experimental applications for SHISA9 antibody with HRP conjugation?

SHISA9 antibody with HRP conjugation serves multiple critical experimental applications in neuroscience research. In immunohistochemistry and immunocytochemistry, it enables precise localization of SHISA9 in brain sections or cultured neurons, particularly at postsynaptic densities. For western blotting, the HRP conjugation provides sensitive detection of SHISA9 protein levels across different brain regions or experimental conditions . Co-immunoprecipitation experiments with HRP-conjugated SHISA9 antibody allow researchers to isolate and identify protein complexes containing SHISA9 and its interacting partners such as PSD95, PSD93, PICK1, GRIP1, and Lin7b . Additionally, the antibody can be used in ELISA assays to quantify SHISA9 levels in tissue homogenates or cerebrospinal fluid samples. Notably, HRP-conjugated antibodies eliminate the need for secondary antibody incubation steps, reducing protocol time and potential sources of background signal .

What are the optimal protocols for conjugating SHISA9 antibody with HRP?

The optimal protocol for conjugating SHISA9 antibody with HRP involves several critical steps to ensure high conjugation efficiency while maintaining antibody functionality. First, start with highly purified IgG-type SHISA9 antibody (>90% pure) at a concentration of approximately 1 mg per reaction . The antibody must be converted to a thiol-antibody using kit components before reaction with maleimide-activated HRP . The maleimide chemistry allows for site-specific conjugation through thiol groups, preventing random cross-linking that might occur with EDC or homobifunctional linkers . Maintain a controlled pH environment (typically pH 7.2-7.4) throughout the conjugation process to prevent antibody denaturation. After conjugation, thorough purification using filtration and scavenger-type purification steps is essential to remove unreacted components, aiming for >90% purity of the final conjugate . Optimize buffer composition (avoiding sodium azide which inhibits HRP activity) and storage conditions (4°C in the short term, aliquoted and frozen at -20°C for long-term storage) to maintain enzymatic activity.

What controls should be included when using SHISA9 antibody in protein interaction studies?

When investigating SHISA9 protein interactions, several controls are essential to ensure experimental validity. First, include a negative control using non-specific IgG matching the host species of the SHISA9 antibody to assess non-specific binding . Second, perform parallel experiments with a SHISA9ΔEVTV mutant (lacking the PDZ-binding motif) to differentiate between specific PDZ domain-mediated interactions and non-specific associations . Third, include competitive binding controls using peptide mimetics, such as a TAT-tagged Shisa9 C-terminal peptide, which can disrupt specific interactions like those with PSD95 . When performing co-immunoprecipitations from brain tissue, compare results between different brain regions (e.g., hippocampus versus cortex) to control for region-specific interactions . Additionally, perform reciprocal co-immunoprecipitations using antibodies against putative binding partners (like PSD95) to confirm interactions from both perspectives. Finally, include lysate input controls and IgG-only beads to account for starting material and non-specific binding to the immunoprecipitation matrix .

How do sample preparation methods affect SHISA9 antibody performance in different applications?

Sample preparation significantly impacts SHISA9 antibody performance across different applications. For immunohistochemistry, tissue fixation method and duration are critical - paraformaldehyde fixation (4%) for 24-48 hours typically preserves SHISA9 epitopes while maintaining tissue architecture. For western blotting, the extraction buffer composition is crucial - SHISA9 resides in the postsynaptic density, which is notoriously difficult to solubilize . Using buffers containing 1-2% detergents like DDM (n-Dodecyl β-D-maltoside) improves SHISA9 extraction while preserving protein-protein interactions . For co-immunoprecipitation, mild solubilization conditions are essential to maintain protein complexes - a two-step extraction protocol with increasing detergent concentrations (e.g., 1% followed by 2% DDM) can optimize yield while preserving interactions . Fresh tissue typically provides better results than frozen for interaction studies. Additionally, protease and phosphatase inhibitors must be included in all extraction buffers to prevent degradation and modification of SHISA9 and its binding partners . For recombinant expression systems, mammalian cells like HEK293T are preferred over bacterial systems to ensure proper post-translational modifications of SHISA9 .

How can researchers investigate SHISA9's role in modulating AMPA receptor function?

Investigating SHISA9's role in AMPA receptor modulation requires sophisticated electrophysiological and molecular approaches. Patch-clamp electrophysiology in acute hippocampal slices can measure AMPA receptor-mediated synaptic currents and their kinetics, particularly in the lateral perforant path where SHISA9 effects are pronounced . Researchers should examine current decay times and paired-pulse facilitation, which are specifically altered by SHISA9's interactions through its PDZ domain . For molecular intervention, TAT-tagged mimetic peptides of the SHISA9 C-terminus can disrupt specific interactions with scaffolding proteins like PSD95 without removing the SHISA9 protein itself . This approach allows for precise dissection of interactor-specific effects, as demonstrated by the observation that disrupting SHISA9-PDZ interactions speeds up AMPAR deactivation and reduces paired-pulse facilitation . Additionally, researchers can use heterologous expression systems to reconstitute AMPA receptors with or without SHISA9 variants (wild-type vs. ΔEVTV mutants) to isolate the contribution of PDZ interactions to receptor function . Combining these approaches with high-resolution imaging techniques can further elucidate how SHISA9 influences AMPAR trafficking, clustering, and synaptic stabilization.

What are the most effective techniques for isolating SHISA9 protein complexes from brain tissue?

Isolating intact SHISA9 protein complexes from brain tissue presents significant challenges due to the dense protein network in the postsynaptic density. The most effective approach involves a carefully optimized immunoprecipitation protocol specifically designed for membrane proteins . Begin by homogenizing fresh mouse cortex or hippocampus in a buffer containing 25 mM HEPES/NaOH (pH 7.4), 0.32 M sucrose, and protease inhibitors . Following low-speed centrifugation (1,000 g for 10 minutes) to remove debris, perform ultracentrifugation (100,000 g for 2 hours) to obtain the membrane-enriched P2 fraction . A two-step solubilization process using DDM detergent (first 2%, then 1%) with 45-minute incubations optimizes extraction while preserving interactions . For immunoprecipitation, use 12 μg of anti-Shisa9 antibody per 6 mg of protein, incubating overnight at 4°C with rotation, followed by capture with agarose-protein A/G beads . After thorough washing with decreasing detergent concentrations, elute complexes with SDS sample buffer for analysis . This approach successfully identified PSD95 as an interactor of Shisa9 in both hippocampus and cortex, despite the challenges of the densely packed PSD structure .

How can researchers troubleshoot low signal issues when using HRP-conjugated SHISA9 antibody?

When encountering low signal with HRP-conjugated SHISA9 antibody, researchers should systematically troubleshoot several potential issues. First, verify HRP enzymatic activity using a simple substrate test before performing the full experiment; activity might be compromised if the antibody was improperly stored or if sodium azide was present in buffers . Second, optimize antigen retrieval methods for fixed tissues - SHISA9 epitopes may require stronger retrieval conditions like high-pH buffers or heat-mediated retrieval . Third, increase antibody concentration incrementally or extend incubation time (overnight at 4°C) to allow for complete antigen binding. Fourth, ensure proper blocking (5% BSA often works better than milk for phospho-proteins) to reduce background while allowing specific binding. Fifth, consider signal amplification systems like tyramide signal amplification which can increase sensitivity by depositing additional HRP substrate around the initial binding site. Finally, if using western blotting, modify transfer conditions - SHISA9 is a membrane protein and may require longer transfer times or different membrane types (PVDF typically works better than nitrocellulose for hydrophobic proteins) . If all else fails, consider concentrating the sample through immunoprecipitation before analysis to enrich for SHISA9.

What experimental approaches can reveal the functional significance of SHISA9-PDZ domain interactions?

Multiple complementary approaches can illuminate the functional significance of SHISA9-PDZ domain interactions. First, electrophysiological recordings in hippocampal slices with application of TAT-Shisa9WT peptide (which disrupts PDZ interactions) versus control peptides reveal that these interactions regulate AMPA receptor deactivation kinetics and paired-pulse facilitation . Second, biochemical competition assays using biotinylated Shisa9 peptides coupled to NeutrAvidin beads can quantify the disruption efficiency of various interventions, providing a measure of binding strength . Third, site-directed mutagenesis generating Shisa9ΔEVTV mutants allows comparison of wild-type and interaction-deficient Shisa9 in both heterologous and native systems . Fourth, viral-mediated expression of these mutants in neuronal cultures or in vivo can reveal long-term consequences of disrupting these interactions on synaptic transmission and plasticity. Fifth, super-resolution microscopy with appropriate antibodies can visualize the spatial organization of SHISA9 and its PDZ partners at synapses, potentially revealing organizational principles. Finally, behavioral testing in animals with manipulated SHISA9-PDZ interactions (using viral vectors or peptides) can connect molecular interactions to cognitive function, particularly in hippocampal-dependent learning tasks.

How should researchers design experiments to investigate SHISA9's influence on hippocampal network activity?

Designing experiments to investigate SHISA9's influence on hippocampal network activity requires multiscale approaches spanning molecular to systems levels. At the molecular level, researchers should use TAT-tagged Shisa9 peptides (wild-type vs. ΔEVTV) to specifically disrupt PDZ interactions while preserving other SHISA9 functions . Field potential recordings in hippocampal slices can then measure network responses to various stimulation protocols in both perforant path and Schaffer collateral pathways . To assess network dynamics in vivo, researchers can employ multielectrode arrays implanted in freely behaving animals with virus-mediated SHISA9 manipulations. Analysis should focus on oscillatory patterns (theta, gamma) during various behavioral states, as SHISA9 modulation of AMPA receptor kinetics likely affects network synchronization . Optogenetic approaches can supplement these studies by allowing precise temporal control of specific pathway activation while monitoring network responses with or without SHISA9-PDZ interaction disruption. Calcium imaging in hippocampal slices or in vivo using genetically encoded calcium indicators provides cellular resolution of network activity across large populations of neurons. Finally, computational modeling incorporating the experimentally determined SHISA9 effects on synaptic transmission can predict and explain network-level consequences of these molecular interactions.

What quantitative methods can accurately measure SHISA9 protein levels in different subcellular fractions?

Accurate quantification of SHISA9 across subcellular fractions requires specialized approaches due to its localization in the difficult-to-solubilize postsynaptic density. A systematic subcellular fractionation protocol should separate brain tissue into at least four fractions: crude homogenate, synaptosomal fraction, synaptic plasma membrane, and postsynaptic density . For each fraction, equal protein amounts should be analyzed by western blotting using HRP-conjugated SHISA9 antibody alongside markers of each compartment (e.g., PSD95 for PSD, synaptophysin for presynaptic terminals) . Quantification should use digital imaging systems with a wide dynamic range to ensure linearity of signal. For absolute quantification, include a standard curve of recombinant SHISA9 protein on each blot. Mass spectrometry-based approaches provide higher specificity and potential for absolute quantification - selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) targeting unique SHISA9 peptides can provide sensitive and specific quantification . For relative distribution studies, immunogold electron microscopy with appropriate controls allows precise localization and semiquantitative analysis of SHISA9 distribution within the postsynaptic compartment, particularly in relation to AMPA receptors and interacting partners like PSD95 .