GRIN2B (Ab-1303) Antibody

What is GRIN2B (Ab-1303) Antibody and what epitope does it recognize?

GRIN2B (Ab-1303) Antibody is a rabbit polyclonal antibody specifically designed to target the GRIN2B protein (Glutamate Receptor Ionotropic NMDA 2B). The antibody recognizes a synthetic non-phosphopeptide derived from human GRIN2B around the phosphorylation site of serine 1303 (Q-H-S(p)-Y-D). This specificity makes it particularly valuable for studying the phosphorylation state of GRIN2B at this critical regulatory site . The antibody is developed in rabbit hosts and demonstrates high specificity to the target epitope in both human and mouse samples.

What are the known species reactivity profiles for GRIN2B (Ab-1303) Antibody?

Based on validated testing data, GRIN2B (Ab-1303) Antibody demonstrates confirmed reactivity with human and mouse samples . This cross-species reactivity makes it a versatile tool for comparative studies between human and murine models. While these species have been confirmed, researchers should perform validation experiments when using this antibody with other species or specialized tissue samples to ensure proper reactivity.

What applications has GRIN2B (Ab-1303) Antibody been validated for?

The antibody has been validated for multiple research applications, with primary validation for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) . These validated applications make it suitable for both quantitative protein detection and qualitative visual identification of target proteins in complex samples. Researchers should conduct preliminary optimization experiments when adapting this antibody for other applications like immunohistochemistry, immunoprecipitation, or flow cytometry.

What are the optimal storage conditions for maintaining GRIN2B (Ab-1303) Antibody activity?

For optimal preservation of antibody activity, GRIN2B (Ab-1303) Antibody should be stored at -20°C or -80°C immediately upon receipt . The antibody is provided in a stabilized solution containing phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol . Repeated freeze-thaw cycles should be avoided as they can significantly impact antibody performance. Aliquoting the antibody into single-use volumes before freezing is recommended for projects requiring multiple experiments over time.

What is the recommended protocol for Western blotting with GRIN2B (Ab-1303) Antibody?

For optimal Western blotting results with GRIN2B (Ab-1303) Antibody, the following protocol is recommended:

• Sample Preparation:

  • Extract total protein from tissue/cells using RIPA buffer containing protease and phosphatase inhibitors

  • Quantify protein concentration using Bradford or BCA assay

  • Prepare samples with loading buffer (containing DTT or β-mercaptoethanol)

  • Heat samples at 95°C for 5 minutes

• Gel Electrophoresis and Transfer:

  • Resolve 20-50μg protein on 8-10% SDS-PAGE gel (GRIN2B is approximately 166 kDa)

  • Transfer proteins to PVDF membrane at 100V for 90 minutes in cold transfer buffer

• Immunoblotting:

  • Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with GRIN2B (Ab-1303) Antibody at 1:500-1:2000 dilution in blocking buffer overnight at 4°C

  • Wash membrane 3× with TBST, 10 minutes each

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000-1:10000) for 1 hour at room temperature

  • Wash membrane 3× with TBST, 10 minutes each

  • Develop using enhanced chemiluminescence substrate

• Expected Results:

  • Primary band at approximately 166 kDa corresponding to GRIN2B

  • Phosphorylation-dependent detection may show variable intensity based on cell/tissue treatment

How should researchers optimize ELISA protocols with GRIN2B (Ab-1303) Antibody?

For ELISA applications with GRIN2B (Ab-1303) Antibody, the following optimization strategy is recommended:

• Antibody Titration:

  • Test antibody across a concentration range (1:500 to 1:5000)

  • Determine optimal concentration that provides strongest specific signal with minimal background

• Sample Preparation:

  • For cell/tissue lysates: Use non-denaturing lysis buffer with phosphatase inhibitors

  • For brain tissue: Special consideration for membrane protein extraction efficiency

• Protocol Optimization:

  • Coating: Use 1-10 μg/ml capture antibody (if using as capture antibody)

  • Blocking: 2-5% BSA in PBS to minimize background

  • Detection: When used as detection antibody, dilute in blocking buffer with 0.05% Tween-20

  • Consider sandwich ELISA approach for greater specificity

• Controls:

  • Include lysates from cells with known GRIN2B expression

  • Include phosphorylated and non-phosphorylated peptide controls

How can GRIN2B (Ab-1303) Antibody be used to study NMDA receptor phosphorylation dynamics?

The GRIN2B (Ab-1303) Antibody is specifically designed to recognize the region surrounding the phosphorylation site of serine 1303, making it an excellent tool for studying phosphorylation dynamics of NMDA receptors. Researchers can implement the following advanced methodologies:

• Temporal Phosphorylation Analysis:

  • Stimulate neuronal cultures with glutamate/NMDA at different time points

  • Lyse cells and perform Western blotting with both phospho-specific and total GRIN2B antibodies

  • Calculate phosphorylation ratio to determine temporal activation patterns

• Pathway Inhibition Studies:

  • Pretreat cells with kinase inhibitors (e.g., PKC, CaMKII, or DAPK1 inhibitors)

  • Measure changes in S1303 phosphorylation to determine pathway contributions

  • Note that DAPK1 has been specifically implicated in S1303 phosphorylation, which enhances synaptic NMDA receptor function

• Co-immunoprecipitation with Phosphorylation Analysis:

  • Use the antibody to immunoprecipitate GRIN2B from neuronal lysates

  • Analyze co-precipitating proteins under different phosphorylation conditions

  • Identify phosphorylation-dependent protein interactions

• Calcium Imaging Correlation:

  • Perform calcium imaging of neurons under various stimulation conditions

  • Correlate calcium influx patterns with GRIN2B phosphorylation status

  • Create a temporal relationship map between receptor activation and phosphorylation

What considerations should be made when using GRIN2B (Ab-1303) Antibody in neurodegenerative disease models?

When applying GRIN2B (Ab-1303) Antibody in neurodegenerative disease research, several important considerations should be addressed:

• Model-Specific Validation:

  • Confirm antibody specificity in the particular disease model

  • Compare phosphorylation patterns between wildtype and disease models

  • Consider age-dependent changes in GRIN2B expression and phosphorylation

• Post-translational Modification Interactions:

  • Analyze how disease-associated proteins (e.g., tau in Alzheimer's) affect GRIN2B phosphorylation

  • Research suggests interactions between tau pathology and NMDA receptor function

  • Compare GRIN2B phosphorylation in brain regions with varying levels of pathology

• Microglial Activation Correlation:

  • Recent research indicates relationships between microglial activation and NMDA receptor function

  • Investigate correlation between disease-associated microglial responses and GRIN2B phosphorylation

  • Consider dual labeling experiments with microglial markers and GRIN2B

• Experimental Design Table for Neurodegenerative Studies:

What are common causes of false negative results with GRIN2B (Ab-1303) Antibody and how can they be addressed?

False negative results can occur for several reasons when working with GRIN2B (Ab-1303) Antibody. Here are common issues and their solutions:

• Inefficient Protein Extraction:

  • GRIN2B is a membrane protein that requires effective solubilization

  • Solution: Use stronger lysis buffers containing 1% SDS or 0.5% sodium deoxycholate

  • For brain tissue samples, consider specialized membrane protein extraction kits

• Phosphatase Activity During Sample Preparation:

  • Since the antibody targets a phosphorylation site, endogenous phosphatases can dephosphorylate the epitope

  • Solution: Use comprehensive phosphatase inhibitor cocktails immediately during tissue/cell lysis

  • Keep samples cold throughout all preparation steps

• Epitope Masking Due to Protein Conformation:

  • In certain experimental conditions, the S1303 site may be masked

  • Solution: Test alternative denaturation methods or milder fixation conditions

  • Consider native vs. denatured detection methods

• Antibody Degradation:

  • Improper storage or handling can reduce antibody activity

  • Solution: Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Verify antibody activity using positive control samples known to express phosphorylated GRIN2B

How can researchers distinguish between specific and non-specific signals when using GRIN2B (Ab-1303) Antibody?

Distinguishing specific from non-specific signals is crucial for accurate interpretation of results. The following approaches are recommended:

• Comprehensive Control Panel:

  • Positive control: Samples with known GRIN2B expression (e.g., hippocampal tissue)

  • Negative control: Samples lacking GRIN2B expression or GRIN2B knockout tissue

  • Peptide competition: Pre-incubate antibody with excess immunizing peptide to block specific binding

  • Secondary-only control: Omit primary antibody to identify secondary antibody non-specific binding

• Signal Validation Techniques:

  • Size verification: GRIN2B should appear at approximately 166 kDa

  • Phosphorylation manipulation: Treat samples with phosphatases to eliminate phospho-specific signal

  • Use multiple antibodies: Compare results with other GRIN2B antibodies targeting different epitopes

• Tissue-Specific Background Reduction:

  • For brain tissue, which has high lipid content:

    • Extended blocking times (2-3 hours)

    • Use specialized blocking reagents (e.g., mouse-on-mouse blocking for mouse samples)

    • Optimize detergent concentration in washing buffers

• Signal Quantification Guidelines:

  • Always normalize phospho-GRIN2B signal to total GRIN2B expression

  • Use internal loading controls appropriate for the subcellular fraction being analyzed

  • Perform multiple technical and biological replicates to ensure reproducibility

How can GRIN2B (Ab-1303) Antibody be used to investigate synaptic versus extrasynaptic NMDA receptor populations?

The distribution and phosphorylation state of GRIN2B differs between synaptic and extrasynaptic locations, with important functional implications. To investigate these populations:

• Subcellular Fractionation Approach:

  • Separate brain tissue into synaptic (PSD-enriched) and extrasynaptic membrane fractions

  • Compare phosphorylation status using GRIN2B (Ab-1303) Antibody

  • Protocol for fractionation:

    • Homogenize tissue in 0.32M sucrose buffer with protease/phosphatase inhibitors

    • Centrifuge at low speed to remove nuclei and cell debris

    • Collect synaptosomal fraction through differential centrifugation

    • Extract PSD-enriched fraction using Triton X-100

    • Isolate extrasynaptic membranes from the non-PSD fraction

• Colocalization Imaging:

  • Perform immunofluorescence with GRIN2B (Ab-1303) Antibody and synaptic markers

  • Use markers like Bassoon (presynaptic) and Homer1 (postsynaptic)

  • Quantify colocalization coefficients to determine synaptic vs. extrasynaptic distribution

• Functional Differentiation Studies:

  • Selectively activate synaptic or extrasynaptic receptors through established protocols

  • Analyze phosphorylation changes using the antibody following selective activation

  • Correlate phosphorylation patterns with calcium imaging or electrophysiology data

• Disease Context Analysis:

  • Extrasynaptic GRIN2B, particularly in concert with DAPK1, has been implicated as a central mediator for stroke damage

  • Compare synaptic vs. extrasynaptic phosphorylation in stroke or excitotoxicity models

  • Analyze how phosphorylation at S1303 relates to receptor trafficking between compartments

What methodological considerations are important when investigating GRIN2B phosphorylation in relation to tau pathology?

Recent research suggests important interactions between tau pathology and NMDA receptor function. When investigating this relationship:

• Co-pathology Analysis Protocol:

  • Perform sequential or multiplex immunostaining with:

    • GRIN2B (Ab-1303) Antibody for phosphorylated receptor

    • Phospho-tau antibodies (e.g., AT8 for Ser202/Thr205)

    • Neuronal markers (e.g., NeuN)

  • Quantify spatial relationships between tau pathology and GRIN2B phosphorylation

• Tau-NMDA Receptor Interaction Studies:

  • Use co-immunoprecipitation to assess physical interactions

  • Compare binding patterns in control vs. pathological conditions

  • Correlate interaction strength with phosphorylation status

• Transgenic Model Considerations:

  • For tau models (e.g., PS19 transgenic mice with P301S mutation) :

    • Analyze age-dependent changes in GRIN2B phosphorylation

    • Compare brain regions with varying tau pathology burden

    • Consider genotype-specific differences in NMDA receptor expression/function

• Therapeutic Intervention Assessment:

  • Measure changes in GRIN2B phosphorylation following tau-targeted therapies

  • Assess whether normalizing tau pathology affects receptor phosphorylation

  • Investigate whether NMDA receptor modulation affects tau pathology progression

How might GRIN2B (Ab-1303) Antibody be integrated into single-cell analysis techniques?

Emerging single-cell technologies offer new opportunities for studying GRIN2B phosphorylation at unprecedented resolution:

• Single-Cell Western Blotting:

  • Apply GRIN2B (Ab-1303) Antibody in microfluidic-based single-cell western blots

  • Compare phosphorylation heterogeneity across individual neurons

  • Correlate with functional phenotypes in the same cells

• Mass Cytometry (CyTOF) Integration:

  • Conjugate GRIN2B (Ab-1303) Antibody with rare earth metals

  • Combine with other phospho-specific antibodies and cell type markers

  • Perform high-dimensional analysis of receptor phosphorylation across cell populations

• Spatial Transcriptomics Correlation:

  • Combine GRIN2B phosphorylation detection with spatial transcriptomics

  • Similar to approaches using CosMx technology and specialized RNA panels

  • Correlate phosphorylation status with local transcriptional signatures

• Super-Resolution Microscopy Applications:

  • Utilize the antibody in STORM or PALM super-resolution microscopy

  • Map nanoscale distribution of phosphorylated GRIN2B at synapses

  • Track phosphorylation-dependent changes in receptor nanoclustering

What are potential applications of GRIN2B (Ab-1303) Antibody in stroke and excitotoxicity research?

Given the role of GRIN2B in stroke pathology, the antibody offers valuable research opportunities:

• Time-Course Phosphorylation Analysis in Stroke Models:

  • Perform temporal profiling of S1303 phosphorylation after ischemic insult

  • Compare phosphorylation patterns in penumbra vs. core ischemic regions

  • Correlate with markers of cell death and neuronal damage

• DAPK1-GRIN2B Interaction Studies:

  • Investigate the relationship between DAPK1 activation and GRIN2B S1303 phosphorylation

  • DAPK1 has been identified as phosphorylating GRIN2B at S1303, enhancing synaptic NMDA receptor function

  • Analyze how this interaction changes during ischemic conditions

• Therapeutic Target Validation:

  • Use the antibody to assess effectiveness of NMDA receptor modulators

  • Measure how various interventions affect S1303 phosphorylation post-stroke

  • Correlate phosphorylation changes with functional recovery markers

• Comparative Pathology Analysis:

  • Contrast GRIN2B phosphorylation patterns across:

    • Different stroke models (global vs. focal ischemia)

    • Various excitotoxic insults (glutamate vs. NMDA vs. oxygen-glucose deprivation)

    • Age-dependent vulnerability to excitotoxicity