GRID2IP Antibody, HRP conjugated

What is GRID2IP and why is it of interest in research?

GRID2IP (Glutamate receptor, ionotropic, delta 2-interacting protein 1) is a Purkinje fiber postsynaptic scaffold protein involved in multiple signal transduction pathways in the nervous system. Recent studies have demonstrated that GRID2IP expression is upregulated in colorectal cancer (CRC) patients and associated with poor prognosis, making it a potential biomarker for cancer research and therapeutic targeting. The protein's ability to influence tumor-associated immune cell infiltration suggests important roles in cancer immunology and potential clinical applications beyond its established neurological functions .

What applications are GRID2IP antibodies typically used for?

GRID2IP antibodies are primarily used for ELISA applications, though they have potential applications in other immunological techniques such as Western blotting, immunocytochemistry, and flow cytometry. The HRP-conjugated version is particularly valuable for detection methods that rely on horseradish peroxidase activity, allowing direct visualization without secondary antibody requirements . When studying GRID2IP in colorectal cancer research, these antibodies have been used for immunoblot analysis to evaluate protein expression levels in various cell lines including CCD18, HT29, and SW480 .

What is the significance of HRP conjugation in GRID2IP antibodies?

HRP (Horseradish Peroxidase) conjugation provides direct enzymatic detection capabilities to the GRID2IP antibody, eliminating the need for secondary antibody incubation steps in many protocols. This conjugation enables colorimetric, chemiluminescent, or fluorescent detection depending on the substrate used. The enzyme catalyzes reactions that produce visible signals when appropriate substrates are added, allowing for sensitive detection of GRID2IP in experimental samples. HRP-conjugated antibodies are valuable for techniques requiring high sensitivity such as ELISA, flow cytometry, and immunohistochemistry .

What is the recommended storage protocol for GRID2IP antibody HRP conjugates?

GRID2IP antibody HRP conjugates should be shipped at 4°C, and upon delivery, should be aliquoted and stored at either -20°C or -80°C to maintain optimal activity. Repeated freeze-thaw cycles should be avoided as they can degrade both the antibody and the HRP enzyme. The antibody is typically provided in a storage buffer containing 0.03% Proclin 300 (as a preservative), 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during storage .

How should researchers validate the specificity of GRID2IP HRP-conjugated antibodies in their experimental systems?

Researchers should implement a multi-faceted validation approach for GRID2IP HRP-conjugated antibodies. First, positive and negative control samples should be included—cell lines known to express GRID2IP (such as HT29 or SW480 for colorectal cancer studies) versus those with minimal expression. Second, competitive inhibition assays using the immunogen peptide (recombinant human Delphilin protein, specifically amino acids 35-156) can confirm binding specificity. Third, siRNA knockdown of GRID2IP in positive control samples should result in reduced signal. Additionally, researchers should perform parallel detection with alternative GRID2IP antibodies from different clones or manufacturers. Western blot analysis should reveal a band corresponding to the expected molecular weight of GRID2IP, and immunohistochemistry should show the expected subcellular localization in Purkinje cells or cancer tissues .

What optimization steps are recommended for GRID2IP antibody use in flow cytometry?

For flow cytometry applications using GRID2IP HRP-conjugated antibodies, researchers should begin with a titration experiment to determine optimal antibody concentration, typically testing dilutions ranging from 1:50 to 1:500. Cell fixation and permeabilization protocols require careful optimization since GRID2IP is primarily intracellular. Paraformaldehyde (2-4%) fixation followed by saponin or methanol permeabilization is often effective. An isotype control (rabbit IgG-HRP with matching concentration) is essential to establish background signal levels. For multi-parameter analysis, researchers should perform compensation controls and use fluorochrome-conjugated tyramide amplification systems compatible with the HRP enzyme. Sample preparation should include proper blocking steps (5-10% serum in PBS) to minimize non-specific binding. Evaluation of both positive populations (immune cells, myofibroblasts) and negative populations can confirm staining specificity .

What are the critical factors affecting sensitivity in GRID2IP detection using HRP-conjugated antibodies?

Several critical factors influence detection sensitivity for GRID2IP using HRP-conjugated antibodies. First, antibody quality and concentration significantly impact results—the >95% purity of Protein G-purified GRID2IP antibodies ensures minimal non-specific binding. Second, substrate selection is crucial; enhanced chemiluminescent reagents (such as those from Thermo Fisher Scientific) provide higher sensitivity than colorimetric substrates for Western blotting applications. Third, incubation conditions affect binding kinetics—overnight incubation at 4°C typically yields better results than shorter incubations at room temperature. Fourth, blocking protocol optimization (using 5% skim milk or alternative blocking agents) minimizes background. Fifth, sample preparation techniques significantly impact epitope accessibility; overly harsh lysis buffers may denature the target epitope region (amino acids 35-156 of human GRID2IP). Finally, signal amplification systems can enhance sensitivity for low-abundance targets. Researchers should systematically optimize each of these parameters for their specific experimental system .

How can GRID2IP antibodies contribute to understanding the protein's role in colorectal cancer progression?

GRID2IP antibodies can be instrumental in elucidating this protein's role in colorectal cancer through multiple advanced approaches. Researchers can perform immunohistochemistry on patient-derived tissue microarrays to correlate GRID2IP expression with clinicopathological features and patient outcomes. Chromatin immunoprecipitation (ChIP) assays combining GRID2IP antibodies with regulatory proteins can reveal transcriptional mechanisms controlling expression. In vitro studies using CCD18, HT29, and SW480 cell lines with differential GRID2IP expression provide models for functional studies, where GRID2IP antibodies can monitor expression following genetic manipulation. Co-immunoprecipitation with GRID2IP antibodies can identify novel protein interaction partners in cancer contexts, potentially revealing cancer-specific signaling networks. Recent findings showing GRID2IP upregulation in colorectal cancer and its association with reduced immune cell infiltration and poor prognosis highlight the importance of these investigations. Particularly valuable is the use of GRID2IP antibodies in single-cell analysis, which has revealed predominant expression in immune cells, myofibroblasts, and cancer cells—providing critical insights into the cellular distribution of this potential biomarker .

What methodological approaches can resolve contradictory results when using GRID2IP antibodies across different sample types?

When facing contradictory results with GRID2IP antibodies across different sample types, researchers should implement a systematic troubleshooting approach. First, epitope mapping analysis should confirm whether different sample processing methods might affect the accessibility of the target epitope (amino acids 35-156 of human GRID2IP). Second, parallel analysis using alternative GRID2IP antibodies targeting different epitopes can determine if the contradictions are antibody-specific or represent true biological variations. Third, quantitative PCR for GRID2IP mRNA should be performed alongside protein detection to determine if discrepancies occur at transcriptional or post-transcriptional levels. Fourth, consideration of sample-specific factors is essential—differences in cancer versus normal tissue may reflect genuine biological heterogeneity rather than methodological issues. Fifth, cross-validation using orthogonal techniques (e.g., mass spectrometry) can provide antibody-independent confirmation. Finally, comprehensive documentation of all experimental variables including fixation methods, incubation times, and sample origins is crucial for identifying the source of contradictions. The growing evidence of GRID2IP expression variability across different cell types (immune cells, myofibroblasts, cancer cells) in colorectal cancer research emphasizes the importance of these considerations .

How can single-cell analysis techniques be combined with GRID2IP antibody detection to advance cancer research?

Single-cell analysis with GRID2IP antibodies represents a cutting-edge approach for cancer research. Researchers can implement single-cell mass cytometry (CyTOF) using metal-conjugated GRID2IP antibodies alongside markers for cell lineage, activation status, and functional characteristics. This enables high-dimensional profiling of GRID2IP expression within tumor microenvironments at unprecedented resolution. Single-cell RNA sequencing paired with protein detection (CITE-seq) allows correlation between GRID2IP transcription and protein expression at the individual cell level. Imaging mass cytometry combines tissue architecture preservation with single-cell resolution, revealing spatial relationships between GRID2IP-expressing cells and other components of the tumor microenvironment. As demonstrated in recent studies using the TISCH database, GRID2IP shows differential expression across immune cells, myofibroblasts, and cancer cells in colorectal tumors, with significant implications for tumor-associated immune cell infiltration. These advanced single-cell approaches enable researchers to characterize heterogeneous expression patterns within tumors, potentially identifying subpopulations of cells where GRID2IP expression correlates with specific functional states or prognostic outcomes .

What signaling pathways interact with GRID2IP in neurological versus oncological contexts?

GRID2IP (Delphilin) serves distinctly different functions in neurological and oncological contexts through interactions with various signaling pathways. In the neurological system, GRID2IP primarily functions as a postsynaptic scaffolding protein at parallel fiber-Purkinje cell synapses, where it links the glutamate receptor GluD2 (GRID2) with the actin cytoskeleton. This interaction facilitates synaptic transmission and plasticity through regulation of receptor clustering and cytoskeletal organization. In contrast, recent oncological research has revealed that GRID2IP influences immune-related pathways in colorectal cancer. Gene Set Enrichment Analysis (GSEA) and single-sample GSEA (ssGSEA) have shown that elevated GRID2IP expression correlates with inhibited infiltration of tumor-associated immune cells, resulting in lower immune scores. This suggests GRID2IP may modulate immune surveillance mechanisms in the tumor microenvironment. Additionally, differential expression analysis between high and low GRID2IP-expressing tumors has identified numerous genes involved in cancer progression and immune response, pointing to GRID2IP's potential role in regulating multiple oncogenic pathways simultaneously .

What experimental designs can best elucidate the functional mechanisms of GRID2IP in cancer progression?

To comprehensively investigate GRID2IP's functional mechanisms in cancer progression, researchers should implement multi-modal experimental approaches. CRISPR-Cas9 gene editing to create GRID2IP knockout and overexpression models in colorectal cancer cell lines (HT29, SW480) allows direct assessment of its impact on proliferation, migration, and invasion. These genetic models should be characterized using GRID2IP antibodies to confirm manipulation success. In vivo xenograft studies with these modified cell lines can evaluate effects on tumor growth, metastasis, and immune infiltration. Patient-derived organoids with varying GRID2IP expression levels provide more physiologically relevant models for drug response studies. Co-culture systems combining cancer cells with immune components can dissect GRID2IP's influence on immune cell recruitment and function. Phosphoproteomic and interactome analyses using immunoprecipitation with GRID2IP antibodies followed by mass spectrometry can identify key interaction partners and post-translational modifications. Chromatin immunoprecipitation sequencing (ChIP-seq) can reveal GRID2IP's potential role in transcriptional regulation. This integrative approach would provide mechanistic insights into how GRID2IP influences both cancer cell-intrinsic properties and immune microenvironment interactions, potentially explaining the observed correlation between high GRID2IP expression and poor prognosis in colorectal cancer patients .

What strategies can overcome cross-reactivity issues when using GRID2IP antibodies in complex tissue samples?

To minimize cross-reactivity issues with GRID2IP antibodies in complex tissue samples, researchers should implement a comprehensive optimization strategy. Pre-adsorption of the antibody with related proteins or tissue lysates known to contain potentially cross-reactive epitopes can remove non-specific antibodies from the polyclonal mixture. Increasing blocking stringency by using a combination of serum proteins, BSA, and non-ionic detergents can reduce non-specific binding. Titration experiments to determine the minimum effective antibody concentration minimize background while maintaining specific signal. Antigen retrieval methods should be carefully optimized for each tissue type, as excessive retrieval can expose cross-reactive epitopes. For the specific GRID2IP antibody developed against human recombinant protein (amino acids 35-156), sequence comparison with potential cross-reactive proteins should guide interpretation of results in non-human samples. When working with human tissue samples containing both neural and cancer components, sequential immunostaining with cell-type specific markers can differentiate GRID2IP expression in different cellular compartments. Peptide competition assays using the specific immunogen fragment provide the gold standard for confirming signal specificity. These approaches are particularly important when studying GRID2IP in colorectal cancer samples, where the protein's expression in multiple cell types (immune cells, myofibroblasts, cancer cells) creates a complex detection environment .

How can researchers troubleshoot weak or inconsistent signals when using GRID2IP HRP-conjugated antibodies?

When encountering weak or inconsistent signals with GRID2IP HRP-conjugated antibodies, researchers should systematically address potential technical and biological factors. First, verify antibody viability by testing HRP enzymatic activity with a direct substrate test; degraded HRP enzymes may require fresh antibody aliquots. Second, optimize antigen retrieval for the specific GRID2IP epitope (amino acids 35-156), testing both heat-mediated and enzymatic methods. Third, enhance detection sensitivity using amplification systems like tyramide signal amplification, which can increase signal by 10-100 fold. Fourth, extend primary antibody incubation time (overnight at 4°C) and optimize concentration through careful titration. Fifth, consider buffer composition adjustments, particularly pH and ionic strength, which can significantly impact antibody-antigen binding kinetics. Sixth, verify sample integrity and GRID2IP expression levels through parallel RNA analysis or alternative antibodies. For colorectal cancer samples with heterogeneous GRID2IP expression across immune cells, myofibroblasts, and cancer cells, consider laser capture microdissection to isolate specific cell populations before analysis. Inconsistent results might reflect genuine biological variability rather than technical issues, particularly given GRID2IP's differential expression patterns across tumor microenvironments .

What are the optimal fixation and permeabilization protocols for GRID2IP detection in different sample types?

Optimization of fixation and permeabilization for GRID2IP detection requires sample-specific approaches. For cultured cells (such as HT29 or SW480 colorectal cancer lines), 4% paraformaldehyde fixation for 15 minutes at room temperature followed by 0.1% Triton X-100 permeabilization for 10 minutes typically preserves GRID2IP epitopes while allowing antibody access. Frozen tissue sections benefit from a brief 10-minute fixation in cold acetone or 4% paraformaldehyde, with minimal permeabilization requirements. Formalin-fixed paraffin-embedded (FFPE) tissues require optimized antigen retrieval—citrate buffer (pH 6.0) heat-induced epitope retrieval often works well for GRID2IP detection, though tris-EDTA (pH 9.0) may yield better results in some tissue types. For flow cytometry applications with cell suspensions, a fixation/permeabilization kit specifically designed for intracellular proteins is recommended. The postsynaptic localization of GRID2IP in neuronal tissues may require specialized permeabilization protocols to access synaptic densities. For immunoblot analysis of GRID2IP in cell lines such as CCD18, HT29, and SW480, NP40 lysis buffer containing protease inhibitors has been successfully used. Researchers should verify protein integrity post-fixation by comparing multiple epitopes or using alternative detection methods. These optimizations are particularly important given GRID2IP's expression in diverse cell types within the colorectal cancer microenvironment, including immune cells, myofibroblasts, and cancer cells .

How might GRID2IP antibodies be incorporated into multiplex immunoassays for cancer biomarker panels?

GRID2IP antibodies can be strategically incorporated into multiplex cancer biomarker panels through several advanced approaches. Metal-tagged GRID2IP antibodies can be included in mass cytometry (CyTOF) panels alongside established colorectal cancer markers and immune cell identifiers, enabling simultaneous quantification of up to 40 proteins at the single-cell level. For tissue-based multiplex immunohistochemistry, GRID2IP antibodies can be included in cyclic immunofluorescence protocols, where sequential staining rounds permit combination with markers of tumor progression, immune infiltration, and signaling pathway activation. Digital spatial profiling platforms allow GRID2IP antibodies to be combined with region-specific analysis of gene expression, creating integrated proteomic and transcriptomic profiles with spatial context. Multiplex bead-based assays can incorporate GRID2IP detection alongside soluble immune mediators in patient serum samples, potentially revealing systemic correlates of tumor GRID2IP expression. Importantly, when developing these multiplex assays, researchers must carefully validate GRID2IP antibody performance in multiplex conditions, as antibody cross-reactivity and interference can occur differently than in single-marker applications. Recent research demonstrating GRID2IP's associations with immune cell infiltration and poor prognosis in colorectal cancer makes it a valuable addition to comprehensive biomarker panels aiming to predict patient outcomes and therapeutic responses .

How can researchers leverage GRID2IP antibodies to investigate the relationship between neurological function and cancer pathology?

Investigating the nexus between GRID2IP's neurological functions and its role in cancer pathology requires sophisticated experimental approaches utilizing specialized antibodies. Researchers can conduct comparative immunohistochemistry studies using identical GRID2IP antibody protocols on both neural tissues (focusing on Purkinje cell synapses) and colorectal cancer specimens to identify shared versus tissue-specific protein interactions and modifications. Brain organoid and cancer organoid models can be developed in parallel, with GRID2IP antibody staining revealing differences in subcellular localization and co-localization patterns. Co-immunoprecipitation experiments using GRID2IP antibodies in both neural and cancer contexts can identify tissue-specific interaction partners, potentially revealing how a synaptic scaffolding protein acquires oncogenic functions. For mechanistic studies, CRISPR-engineered mutations affecting specific GRID2IP domains can determine which structural elements are essential for its neural versus cancer-related functions. Particularly intriguing is examining whether GRID2IP's ability to link GRID2 with the actin cytoskeleton in neurons is repurposed in cancer cells to influence migration and invasion capabilities. Single-cell multiomics approaches combining GRID2IP protein detection with transcriptomics can identify cell states where neural-like signaling pathways are activated in tumors. This cross-disciplinary research has significant implications, as proteins functioning at the neural-cancer interface could reveal fundamental biological mechanisms and identify novel therapeutic targets with established CNS safety profiles .