Phospho-GRIN2B (Tyr1474) Antibody

What is GRIN2B and what is the significance of its phosphorylation at Tyr1474?

GRIN2B (also known as GluN2B) is a subunit of N-methyl-D-aspartate (NMDA) receptors, which are ionotropic glutamate receptors that play crucial roles in synaptic transmission, synaptic plasticity, and neurodevelopment . The gene encoding this protein, GRIN2B, has been implicated in various neurodevelopmental disorders, including intellectual disability, developmental delay, and epilepsy .

Phosphorylation at Tyr1474 is a critical post-translational modification that stabilizes synaptic NMDAR on the cell surface by preventing the interaction of the clathrin adaptor protein with the YEKL motif, thereby inhibiting endocytosis . This regulation of surface expression directly impacts receptor function and signaling capabilities in neurons.

What are the primary research applications for Phospho-GRIN2B (Tyr1474) Antibody?

Phospho-GRIN2B (Tyr1474) Antibody has several key applications in neuroscience and cell signaling research:

How does GRIN2B phosphorylation differ from other NMDA receptor modifications?

GRIN2B phosphorylation at Tyr1474 specifically regulates receptor trafficking and surface expression, while phosphorylation at other sites serves different functions. For instance, phosphorylation at Tyr1252 fosters binding to the actin-regulatory protein Nck2, enhancing NMDAR functions related to cytoskeletal organization . Additionally, phosphorylation at Ser1303 by DAPK1 enhances synaptic NMDA receptor channel activity .

Unlike some other post-translational modifications, tyrosine phosphorylation of GRIN2B at residue 1474 directly affects receptor internalization processes rather than channel gating properties, positioning it as a critical regulator of receptor availability at synaptic sites .

What are the recommended protocols for using Phospho-GRIN2B (Tyr1474) Antibody in Western blot experiments?

For optimal Western blot results when using Phospho-GRIN2B (Tyr1474) Antibody:

• Sample Preparation:

  • Lyse cells/tissues in RIPA buffer containing phosphatase inhibitors to preserve phosphorylation status

  • Denature proteins in Laemmli buffer at 95°C for 5 minutes

  • Load 20-50μg of total protein per well on SDS-PAGE gel

• Electrophoresis and Transfer:

  • Use 6-8% gels due to the large size of GRIN2B (~180-200 kDa)

  • Perform wet transfer to PVDF membrane at 30V overnight at 4°C for efficient transfer of large proteins

• Antibody Incubation:

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Incubate with Phospho-GRIN2B (Tyr1474) antibody at 1:500-1:2000 dilution overnight at 4°C

  • Wash extensively with TBST (3-5 times, 5-10 minutes each)

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

• Validation Controls:

  • Include lambda phosphatase-treated samples as negative controls

  • The observed molecular weight should be approximately 200 kDa

What are effective strategies for optimizing immunohistochemistry with Phospho-GRIN2B (Tyr1474) Antibody?

To achieve optimal immunohistochemistry results:

• Tissue Preparation:

  • Fix tissues in 4% paraformaldehyde

  • Perform antigen retrieval by boiling sections in citric acid buffer (pH 6.0) for 1 hour at 95°C

• Blocking and Antibody Incubation:

  • Block with 5% normal goat serum in TBS-Tween-Triton for 1 hour

  • Incubate with primary antibody at 1:100-1:300 dilution overnight at 4°C

  • Use HRP-conjugated anti-rabbit secondary antibody for 1 hour at room temperature

  • Develop with DAB and counterstain nuclei with hematoxylin

• Critical Parameters:

  • Maintain consistent timing for all samples to ensure comparative analysis

  • Include both positive controls (tissues known to express phosphorylated GRIN2B) and negative controls (phosphatase-treated sections)

  • For fluorescence detection, use Alexa Fluor 488-conjugated anti-rabbit secondary antibody and analyze with confocal microscopy

How can I verify the specificity of Phospho-GRIN2B (Tyr1474) Antibody in my experiments?

Several approaches can confirm antibody specificity:

• Phosphatase Treatment: Treat duplicate samples with lambda phosphatase to remove phosphorylation. The signal should be significantly reduced or eliminated in treated samples, as demonstrated in studies with similar phospho-specific antibodies .

• Peptide Competition: Pre-incubate the antibody with the phosphorylated peptide immunogen (typically containing the sequence around Tyr1474). This should block specific binding and reduce signal.

• Kinase Activation/Inhibition: Treat cells with agents known to modulate GRIN2B phosphorylation. For example, HGF treatment increases GRIN2B phosphorylation, while treatment with specific kinase inhibitors should reduce it .

• Genetic Controls: Use GRIN2B knockout cells/tissues or cells expressing a Y1474F mutant that cannot be phosphorylated at this site as negative controls.

How does phosphorylation of GRIN2B at Tyr1474 contribute to synaptic function and neurodevelopmental disorders?

Phosphorylation of GRIN2B at Tyr1474 plays a crucial role in synaptic function by:

• Regulating Receptor Trafficking: Phosphorylation at Tyr1474 stabilizes NMDARs at the synaptic membrane by preventing endocytosis, thus maintaining the receptor population available for signaling .

• Impact on Neuronal Development: GRIN2B is predominantly expressed during early postnatal development , and its proper regulation through phosphorylation is critical for synaptogenesis and neuronal circuit formation. Mutations in GRIN2B are associated with neurodevelopmental disorders characterized by intellectual disability, developmental delay, and epilepsy .

• Synaptic Plasticity Regulation: By controlling receptor abundance at synapses, phosphorylation at Tyr1474 influences long-term potentiation and depression, which are cellular mechanisms underlying learning and memory.

Dysregulation of this phosphorylation process may contribute to the pathophysiology of GRIN2B-related neurodevelopmental disorders by:

• Altering synaptic NMDAR levels and signaling capacity

• Disturbing the balance between receptor insertion and internalization

• Affecting downstream signaling pathways that regulate dendritic spine formation and synaptic strength

What experimental approaches can effectively assess the functional consequences of GRIN2B phosphorylation at Tyr1474?

Several advanced techniques can evaluate the functional impact of GRIN2B phosphorylation:

• Electrophysiological Recordings:

  • Whole-cell patch-clamp recordings to measure NMDAR-mediated currents

  • Analysis of evoked EPSCs in neurons expressing wild-type versus phospho-mutant GRIN2B

  • Comparison of receptor desensitization and recovery kinetics

• Advanced Imaging Techniques:

  • Live-cell imaging with pH-sensitive GFP-tagged receptors to track surface expression dynamics

  • FRAP (Fluorescence Recovery After Photobleaching) to measure receptor mobility

  • Super-resolution microscopy to analyze nanoscale receptor clustering

• Molecular Interaction Studies:

  • Proximity Ligation Assay to detect interactions between GRIN2B and binding partners

  • Co-immunoprecipitation to identify phosphorylation-dependent protein complexes

  • BRET/FRET assays to measure real-time protein interactions in living cells

• Functional Readouts:

  • Analysis of calcium influx using calcium-sensitive dyes or genetically encoded calcium indicators

  • Assessment of downstream signaling pathway activation (e.g., CaMKII, CREB phosphorylation)

  • Evaluation of synaptic plasticity through LTP/LTD induction protocols

How do GRIN2B mutations affect phosphorylation at Tyr1474 and subsequent receptor function?

Disease-causing mutations in GRIN2B can impact phosphorylation at Tyr1474 through several mechanisms:

What are the critical factors for preserving and accurately detecting GRIN2B phosphorylation status?

Preserving phosphorylation status is crucial for accurate analysis:

• Sample Collection and Preservation:

  • Rapid sample collection and processing to minimize post-mortem dephosphorylation

  • Immediate addition of phosphatase inhibitor cocktails to all buffers

  • Flash-freezing samples in liquid nitrogen when immediate processing is not possible

• Lysis Conditions:

  • Use cold lysis buffers containing sodium orthovanadate (1-2 mM) to inhibit tyrosine phosphatases

  • Include phosphatase inhibitors like sodium fluoride (10 mM) and beta-glycerophosphate (5-10 mM)

  • Maintain samples at 4°C throughout processing

• Handling Considerations:

  • Avoid repeated freeze-thaw cycles of antibodies and samples

  • Store antibodies in small aliquots at -20°C

  • Prepare fresh working solutions of all reagents for each experiment

• Detection Optimization:

  • Use PVDF rather than nitrocellulose membranes for Western blotting of large phosphoproteins

  • Consider using signal enhancement systems for low-abundance phosphoproteins

  • Validate results using multiple detection methods (e.g., Western blot and immunofluorescence)

What approaches are most effective for studying the temporal dynamics of GRIN2B phosphorylation?

To investigate temporal phosphorylation dynamics:

• Time-Course Stimulation Experiments:

  • Treat cells with agonists (e.g., glutamate, NMDA) or growth factors (e.g., HGF) for varying durations

  • Collect samples at multiple timepoints (e.g., 0, 5, 15, 30, 60 minutes)

  • Process all samples simultaneously to ensure comparable conditions

• Pulse-Chase Analysis:

  • Label surface receptors with biotin or antibodies against extracellular epitopes

  • Allow internalization to occur over various time periods

  • Analyze remaining surface receptors versus internalized pool to correlate with phosphorylation status

• Live-Cell Imaging:

  • Utilize phospho-sensitive fluorescent biosensors based on phospho-binding domains

  • Perform real-time imaging during receptor activation and signaling

  • Correlate phosphorylation events with receptor clustering and mobility

• Computational Modeling:

  • Develop kinetic models of GRIN2B phosphorylation and trafficking

  • Integrate experimental data to predict temporal patterns of phosphorylation

  • Validate model predictions with targeted experiments

How can I analyze the interplay between GRIN2B phosphorylation at Tyr1474 and other post-translational modifications?

Investigating cross-talk between different modifications requires specialized approaches:

• Multi-Antibody Analysis:

  • Perform sequential or parallel detection with antibodies against different modifications

  • Strip and reprobe Western blots to detect multiple modifications on the same sample

  • Use differentially labeled secondary antibodies for simultaneous detection of multiple modifications

• Mass Spectrometry:

  • Conduct phosphoproteomic analysis to identify all phosphorylation sites on GRIN2B

  • Use targeted MS approaches to quantify specific phosphorylation combinations

  • Perform temporal analysis to determine sequential modification patterns

• Proximity Ligation Assay Adaptations:

  • Use antibody pairs targeting different modifications to detect co-occurrence

  • Quantify signals to determine the proportion of receptors with multiple modifications

  • Compare modification patterns under different stimulation conditions

• Mutation Analysis:

  • Generate phospho-mimetic or phospho-deficient mutations at multiple sites

  • Assess how mutation of one site affects phosphorylation at other sites

  • Evaluate functional consequences of combined mutations

What are the most common problems when working with Phospho-GRIN2B (Tyr1474) Antibody and how can they be resolved?

How can I optimize detection of phosphorylated GRIN2B in tissues with low expression levels?

For tissues with low GRIN2B phosphorylation:

• Sample Enrichment Techniques:

  • Perform immunoprecipitation with total GRIN2B antibody before probing for phosphorylation

  • Use phospho-tyrosine antibodies for initial enrichment followed by GRIN2B detection

  • Consider subcellular fractionation to concentrate postsynaptic density proteins

• Signal Amplification Methods:

  • Utilize tyramide signal amplification for IHC/IF applications

  • For Western blots, use high-sensitivity ECL substrates or fluorescent detection systems

  • Consider biotin-streptavidin amplification systems for ELISA formats

• Alternative Detection Approaches:

  • Try Proximity Ligation Assay for enhanced sensitivity in detecting phosphorylated proteins

  • Consider using Cell-Based ELISA kits specifically designed for phospho-protein detection

  • Explore mass spectrometry-based targeted approaches for absolute quantification

• Experimental Design Considerations:

  • Include positive controls from tissues/cells with known high phosphorylation levels

  • Use stimulation protocols known to enhance GRIN2B phosphorylation (e.g., HGF treatment)

  • Pool multiple samples when possible to increase starting material

By implementing these specialized techniques and considerations, researchers can effectively detect and analyze GRIN2B phosphorylation even in challenging experimental contexts.

How does GRIN2B phosphorylation at Tyr1474 relate to its role in non-neuronal tissues and pathological conditions?

While GRIN2B is primarily studied in neurons, emerging research has identified important roles in other contexts:

• Cancer Biology:

  • GRIN2B phosphorylation has been implicated in MET oncogene-driven breast cancer invasion

  • HGF stimulation leads to phosphorylation of GRIN2B at Tyr1474 in triple-negative breast cancer cells

  • The phosphorylation stabilizes interaction between MET and NMDAR, potentially enhancing invasive capabilities

• Glial Function:

  • While GluN2B is not as strongly expressed in astrocytes as other NMDAR subunits, NMDARs have been observed in astrocytes and activated microglia

  • The regulation of GRIN2B phosphorylation in these cell types remains poorly understood

  • Phosphorylation status may impact neuron-glia communication and neuroinflammatory processes

• Therapeutic Implications:

  • Understanding phosphorylation mechanisms may help develop more targeted approaches for treating GRIN2B-related disorders

  • Kinase inhibitors targeting the enzymes responsible for Tyr1474 phosphorylation could have therapeutic potential

  • Pharmacological inhibition of NMDAR leads to impairment of cell migration and invasion in cancer, suggesting potential for combination therapies

What are emerging technologies for studying GRIN2B phosphorylation at the single-cell or subcellular level?

Cutting-edge approaches for high-resolution analysis include:

• Advanced Imaging Technologies:

  • Super-resolution microscopy (STORM, PALM, STED) to visualize nanoscale receptor clustering and phosphorylation patterns

  • Expansion microscopy to physically enlarge specimens for improved resolution of phospho-protein localization

  • Lattice light-sheet microscopy for rapid 3D imaging of phosphorylation dynamics in living cells

• Single-Cell Analysis:

  • Phospho-flow cytometry for quantitative analysis of GRIN2B phosphorylation at the single-cell level

  • Single-cell Western blotting for heterogeneity assessment in phosphorylation status

  • Mass cytometry (CyTOF) with phospho-specific antibodies for multiparametric analysis

• Subcellular Resolution Techniques:

  • Optogenetic control of kinases/phosphatases for spatiotemporally precise manipulation

  • Genetically encoded FRET-based phosphorylation sensors targeted to specific subcellular compartments

  • Correlative light and electron microscopy to link phosphorylation status to ultrastructural features

• Molecular Engineering Approaches:

  • CRISPR-based tagging of endogenous GRIN2B for live monitoring of phosphorylation

  • Engineered phospho-binding domains linked to fluorescent proteins for real-time visualization

  • Photocaged phospho-amino acids for precise temporal control of phosphorylation states