GRIN2A Antibody

What is GRIN2A and why is it significant in neuroscience research?

GRIN2A encodes the glutamate ionotropic receptor NMDA type subunit 2A in humans, also known by alternative names including GluN2A, NMDAR2A, NR2A, EPND, FESD, and glutamate receptor ionotropic NMDA 2A. This protein is a crucial subunit of NMDA receptors with a molecular weight of approximately 165.3 kilodaltons . GRIN2A has gained significant research attention due to its established association with epilepsy-aphasia syndromes, neurodevelopmental disorders, and schizophrenia. Mutations in this gene can result in both gain-of-function and loss-of-function effects, contributing to various neurological phenotypes . Research into GRIN2A is critical for understanding the molecular basis of these conditions and developing potential therapeutic approaches.

Which species demonstrate conservation of GRIN2A for cross-reactivity studies?

When planning cross-species studies, researchers should note that GRIN2A orthologs have been identified in canine, porcine, monkey, mouse, and rat models . This conservation enables comparative studies across these species using certain antibodies. Many commercial antibodies exhibit reactivity with human, mouse, and rat GRIN2A proteins, making these models particularly valuable for translational research . When selecting antibodies for cross-species applications, researchers should verify the specific reactivity profile of their chosen antibody, as some are species-specific while others offer broader reactivity. Sequence alignment analysis between human GRIN2A and the target species is recommended before experimental design to ensure epitope conservation.

What are the common experimental applications for GRIN2A antibodies?

GRIN2A antibodies are utilized across multiple experimental techniques including Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC), Immunofluorescence (IF), and Flow Cytometry (FCM) . Each application requires specific optimization of antibody concentration and experimental conditions. For instance, immunohistochemistry applications typically use dilutions ranging from 1:20 to 1:200 . Western blotting has been successfully performed at concentrations of 3.2μg/ml with predicted band sizes of 166 and 145 kDa . When planning experiments, researchers should select antibodies validated for their specific application of interest, as performance can vary significantly between techniques even with the same antibody.

How should researchers select between monoclonal and polyclonal GRIN2A antibodies?

The choice between monoclonal and polyclonal GRIN2A antibodies depends on the research objective. Monoclonal antibodies, such as the GRIN2A (3C3) monoclonal antibody listed in the search results, offer high specificity for a single epitope, making them ideal for applications requiring consistent lot-to-lot reproducibility . They are particularly valuable for quantitative studies and when background signals must be minimized. Polyclonal antibodies, by contrast, recognize multiple epitopes on the GRIN2A protein, potentially offering higher sensitivity but with greater batch variation. For novel research applications, polyclonal antibodies may detect the protein of interest even if some epitopes are modified or masked. When studying specific mutations or post-translational modifications, researchers should select antibodies whose epitopes do not overlap with the region of interest.

What storage and handling protocols maximize GRIN2A antibody performance?

Optimal storage conditions for GRIN2A antibodies typically include keeping them at -80°C for long-term storage . Many commercial antibodies are supplied in PBS buffer, sometimes with additional stabilizers. For example, the recombinant GRIN2A antibody (83465-3-PBS) from Proteintech is provided in PBS only (BSA and azide free) at a concentration of 1 mg/mL . During experimental procedures, antibodies should be kept on ice and exposure to repeated freeze-thaw cycles should be minimized. Small aliquots for single use are recommended to preserve antibody integrity. When working with conjugation-ready formats, researchers should follow supplier-specific protocols for the conjugation procedure and subsequent storage of the modified antibody.

What controls are essential when working with GRIN2A antibodies?

Proper experimental controls are critical for GRIN2A antibody applications. Positive controls should include samples known to express GRIN2A, such as U87 cell lysates, rat heart tissue, or mouse liver tissue, which have been verified to show specific bands at the expected molecular weight (166 kDa) . Negative controls should include samples where GRIN2A expression is absent or in tissues from GRIN2A knockout models when available. For immunohistochemistry or immunofluorescence, secondary antibody-only controls are essential to identify non-specific binding. Additionally, pre-absorption controls, where the primary antibody is pre-incubated with the immunizing peptide, can confirm specificity. For quantitative applications, researchers should include a standard curve using recombinant GRIN2A protein at known concentrations.

How can researchers utilize GRIN2A antibodies to characterize disease-associated mutations?

GRIN2A antibodies play a crucial role in characterizing functional consequences of disease-associated mutations. Recent research has identified various GRIN2A variants in patients with epilepsy-aphasia syndrome, such as c.2482A>G/p.M828V and c.2627T>C/p.I876T . Antibody-based techniques like immunofluorescence assays can measure both surface and total expression levels of wild-type versus mutant GluN2A proteins. The methodology involves transfecting cells with GRIN1 and GRIN2A-EGFP plasmids (both wild-type and mutant variants), followed by antibody labeling and confocal microscopy to quantify protein expression and localization . This approach has revealed that certain mutations result in reduced surface expression of the GluN2A subunit, providing critical insights into the mechanistic basis of associated disorders.

What are the key differences between loss-of-function and gain-of-function GRIN2A variants?

GRIN2A mutations can result in either loss-of-function (LoF) or gain-of-function effects, with distinct implications for neurological phenotypes. Recent research has demonstrated that schizophrenia-associated GRIN2A variants predominantly display loss-of-function effects, whereas epilepsy and developmental delay/intellectual disability-associated variants can exhibit both gain- and loss-of-function characteristics . To distinguish between these functional consequences, researchers employ antibody-based techniques in combination with electrophysiological recordings. Typically, cells expressing wild-type or mutant GRIN2A are subjected to whole-cell voltage-clamp current recordings to assess receptor function, while immunofluorescence assays with anti-GRIN2A antibodies evaluate protein expression and trafficking . This multimodal approach enables researchers to correlate molecular alterations with functional outcomes and disease phenotypes.

What are the optimal immunofluorescence protocols for detecting GRIN2A?

Successful immunofluorescence detection of GRIN2A requires careful optimization of protocols. Based on published methodologies, a standard approach involves transfecting cells with GRIN1 and GRIN2A-EGFP plasmids 24 hours prior to the assay . For surface expression analysis, cells are incubated with a primary antibody targeting an extracellular epitope or a tag (such as anti-GFP antibody at 1:1000 dilution) followed by a fluorophore-conjugated secondary antibody (like Alexa Fluor 647 at 1:1000 dilution) . Cells are then fixed with 4% paraformaldehyde for 10 minutes before mounting on glass slides. For total protein detection, cells are first fixed and permeabilized before antibody incubation. Images are captured using confocal microscopy, and fluorescence intensity is quantified using image analysis software such as ImageJ2. This approach allows for the comparison of surface-to-total expression ratios between wild-type and mutant GluN2A proteins.

How can non-specific binding issues be mitigated in GRIN2A immunodetection?

Non-specific binding is a common challenge when working with GRIN2A antibodies. Several strategies can minimize this issue: (1) Optimize blocking conditions by testing different blocking agents (BSA, normal serum, commercial blocking buffers) at various concentrations and incubation times; (2) Titrate antibody concentrations to find the optimal dilution that maximizes specific signal while minimizing background; (3) Increase washing duration and frequency between antibody incubation steps; (4) For Western blotting applications, consider using alternate membrane types or blocking agents if persistent background issues occur; (5) In immunohistochemistry, employ antigen retrieval methods optimized for GRIN2A detection, as improper epitope exposure can contribute to non-specific binding; (6) For immunofluorescence, use Sudan Black B to reduce autofluorescence in fixed tissues. Implementing these approaches systematically can significantly improve signal-to-noise ratios in GRIN2A detection assays.

What factors affect the reproducibility of GRIN2A antibody-based experiments?

Several factors influence the reproducibility of GRIN2A antibody experiments: (1) Antibody source and lot-to-lot variability—recombinant monoclonal antibodies like Proteintech's rabbit recombinant GRIN2A antibody (83465-3-PBS) offer enhanced batch-to-batch consistency compared to traditional antibodies ; (2) Sample preparation methods—variations in protein extraction, fixation, or antigen retrieval can significantly impact results; (3) Experimental conditions including temperature, incubation times, and buffer compositions; (4) Detection methods and instrumentation sensitivity; (5) Cellular context, as GRIN2A expression and localization may vary with cell type, developmental stage, or activity state. To maximize reproducibility, researchers should maintain detailed protocols, use consistent reagent sources, include appropriate controls in each experiment, and validate new antibody lots against previous results before conducting critical experiments.

How are GRIN2A antibodies contributing to the study of NMDA receptor trafficking?

GRIN2A antibodies have become instrumental in investigating NMDA receptor trafficking dynamics. Through immunofluorescence assays, researchers can differentiate between surface-expressed and intracellular pools of GluN2A subunits . This approach involves sequential labeling with anti-GRIN2A antibodies before and after cell permeabilization or using GFP-tagged GRIN2A constructs with anti-GFP antibodies. These techniques have revealed that certain disease-associated mutations, such as p.M828V and p.I876T, result in reduced surface-to-total expression ratios of GluN2A . By combining antibody-based trafficking assays with electrophysiological recordings, researchers can correlate altered receptor localization with functional deficits. Future applications may include live-cell imaging with fluorescently conjugated GRIN2A antibody fragments to track receptor dynamics in real-time, providing deeper insights into the temporal aspects of NMDA receptor trafficking in health and disease states.

How can GRIN2A antibodies be utilized in multiplex assays with other NMDA receptor subunits?

Multiplex analysis of NMDA receptor subunits offers comprehensive insights into receptor composition and stoichiometry in different contexts. GRIN2A antibodies can be incorporated into multiplex assays such as cytometric bead arrays, which allow simultaneous detection of multiple proteins from a single sample . For successful multiplexing, researchers should select antibodies with complementary properties: (1) Choose antibodies raised in different host species or of different isotypes to allow discrimination with species- or isotype-specific secondary antibodies; (2) Select fluorophore combinations with minimal spectral overlap for immunofluorescence applications; (3) For co-immunoprecipitation studies, use antibodies that recognize non-overlapping epitopes to avoid competitive binding; (4) Consider matched antibody pairs, such as Proteintech's MP00461-1 which includes 83465-3-PBS for capture and 83465-4-PBS for detection, specifically validated for cytometric bead array applications . These multiplex approaches enable researchers to investigate the complex interplay between GRIN2A and other NMDAR subunits in different physiological and pathological contexts.