Phospho-GRIN1 (Ser896) Antibody

What is Phospho-GRIN1 (Ser896) Antibody and what epitope does it recognize?

Phospho-GRIN1 (Ser896) Antibody is a rabbit polyclonal antibody that specifically recognizes the NMDA receptor subunit 1 (NMDAR1/GRIN1) only when phosphorylated at serine residue 896. The antibody is generated using synthetic phospho-peptides derived from human NMDAR1 around the phosphorylation site of Ser896 . This antibody is crucial for studying the phosphorylation state of NMDAR, which regulates receptor function in neuronal signaling pathways .

What are the optimal protocols for using Phospho-GRIN1 (Ser896) Antibody in different applications?

For Western Blotting: Use dilutions ranging from 1:500 to 1:2000 , with some manufacturers recommending 1:1000 . Samples should be prepared in denaturing conditions, and transfer to PVDF membranes is typically recommended for phospho-specific antibodies.

For Immunohistochemistry: Recommended dilutions range from 1:50 to 1:300 . Optimal fixation involves 4% paraformaldehyde, and antigen retrieval is often necessary (citrate buffer pH 6.0 is commonly used) to unmask phospho-epitopes.

For ELISA applications: Much higher dilutions are effective, with recommendations ranging from 1:10,000 to 1:40,000 , demonstrating the high sensitivity of this antibody in ELISA formats.

How should researchers optimize sample preparation to preserve phosphorylation at Ser896?

To preserve the phosphorylation state at Ser896, samples must be collected and processed with phosphatase inhibitors present in all buffers. Recommended practices include:

• Immediate flash-freezing of tissue samples in liquid nitrogen

• Homogenization in RIPA or similar buffers containing phosphatase inhibitor cocktails

• Maintaining samples at 4°C throughout processing

• Avoiding multiple freeze-thaw cycles which can reduce phospho-epitope integrity

• Including sodium orthovanadate (1-2 mM) and sodium fluoride (10 mM) as specific phosphatase inhibitors

What controls should be included when using Phospho-GRIN1 (Ser896) Antibody?

Proper experimental design should include:

• Positive control: Samples from cells treated with PKC activators (e.g., PMA) which enhance Ser896 phosphorylation

• Negative control: Samples treated with lambda phosphatase to remove phosphorylation

• Specificity control: Preincubation of the antibody with the immunizing phosphopeptide

• Loading control: Probing for total GRIN1 protein using a phosphorylation-independent antibody

• Transfection control: Comparing wild-type GRIN1 with S896A mutant samples

How does phosphorylation at Ser896 functionally differ from other GRIN1 phosphorylation sites?

Phosphorylation of GRIN1 occurs at multiple sites with distinct functional consequences:

Understanding these differences is crucial for experimental design when studying NMDAR regulation, as differential phosphorylation creates distinct functional states of the receptor that influence synaptic plasticity, learning, and neuroplasticity .

How can researchers investigate the relationship between PKC activation and GRIN1 Ser896 phosphorylation?

To study the relationship between PKC activation and Ser896 phosphorylation, researchers can:

• Perform time-course experiments with PKC activators (PMA, diacylglycerol analogs) and monitor Ser896 phosphorylation

• Use PKC inhibitors (GF109203X, Gö6983) to block phosphorylation

• Compare phosphorylation levels in normal conditions versus during activity-dependent plasticity

• Implement phosphomimetic (S896D/E) or phospho-deficient (S896A) mutants to study functional consequences

• Analyze co-immunoprecipitation of PKC isoforms with NMDAR complexes during receptor activation

These approaches allow researchers to establish causality between PKC signaling pathways and functional changes in NMDAR activity mediated through Ser896 phosphorylation .

What is the significance of GRIN1 Ser896 phosphorylation in synaptic plasticity and learning?

NMDA receptors are critical mediators of long-term potentiation (LTP) and synaptic plasticity underlying learning and memory formation. Phosphorylation at Ser896 specifically:

• Modulates calcium permeability of the NMDAR channel

• Influences receptor trafficking and membrane insertion

• Affects protein-protein interactions with postsynaptic scaffolding molecules

• Regulates calmodulin binding, which normally inhibits NMDAR function

• May serve as a molecular switch during activity-dependent plasticity

Experimental approaches to study these effects include electrophysiological recordings combined with phospho-specific antibody staining, calcium imaging in neurons expressing wild-type versus phospho-mutant receptors, and behavioral assays in animals with altered PKC-mediated phosphorylation pathways .

Why might researchers observe discrepancies in molecular weight between different experiments?

Discrepancies in observed molecular weight between the expected 105 kDa and the frequently observed 120 kDa can result from:

• Different splice variants of GRIN1 with varying molecular weights

• Post-translational modifications (glycosylation, ubiquitination)

• Sample preparation methods (reducing vs. non-reducing conditions)

• Gel percentage and running conditions affecting migration

• Different species sources (human vs. rodent)

To address these discrepancies, researchers should run appropriate molecular weight standards, compare their results with literature values, and consider verifying protein identity through mass spectrometry or additional antibodies targeting different epitopes of GRIN1.

How can researchers optimize antibody detection in tissues with low NMDAR expression?

For tissues or samples with low NMDAR expression levels:

• Implement signal amplification methods such as tyramide signal amplification for immunohistochemistry

• Use highly sensitive detection systems (enhanced chemiluminescence for Western blots)

• Enrich NMDAR by immunoprecipitation before analysis

• Consider tissue-specific extraction buffers optimized for membrane proteins

• Increase protein loading while ensuring the linear range of detection is maintained

• For Western blots, use more sensitive membranes like low-fluorescence PVDF

• Extend primary antibody incubation time to 24-48 hours at 4°C

What strategies can address non-specific binding when using Phospho-GRIN1 (Ser896) Antibody?

When encountering non-specific binding:

• Optimize blocking conditions (5% BSA is generally preferred over milk for phospho-specific antibodies)

• Include phosphatase inhibitors throughout sample preparation

• Increase washing stringency by adding 0.1-0.3% Tween-20 to wash buffers

• Perform antigen preabsorption controls to confirm specificity

• Consider alternative blocking agents like fish gelatin which can reduce background

• Titrate antibody concentration carefully, as higher concentrations may increase non-specific binding

• For immunohistochemistry, include 0.3% Triton X-100 in antibody diluent to improve penetration and reduce non-specific interactions

What are the optimal storage conditions for maintaining Phospho-GRIN1 (Ser896) Antibody activity?

To maintain optimal antibody activity:

• Store concentrated stocks at -20°C in small aliquots to avoid freeze-thaw cycles

• The antibody is typically supplied in PBS with 50% glycerol and stabilizers like 0.02% sodium azide

• Working dilutions should be prepared fresh and can be stored at 4°C for up to one week

• Avoid more than 5 freeze-thaw cycles which can significantly reduce activity

• When stored properly, the antibody should remain stable for approximately one year

How should researchers validate antibody performance after extended storage?

To validate antibody performance after extended storage:

• Run parallel experiments with newly purchased antibody and stored antibody

• Test dilution series to determine if optimal working concentration has changed

• Include positive controls with known phosphorylation status

• Verify signal-to-noise ratio is comparable to previous experiments

• Check for increased background or non-specific binding which may indicate degradation

• If performance has decreased, consider purifying the antibody using antigen affinity methods or purchasing new stock

How does the specificity of different commercially available Phospho-GRIN1 (Ser896) antibodies compare?

Based on the search results, multiple manufacturers offer Phospho-GRIN1 (Ser896) antibodies with similar specifications but some notable differences:

Researchers should select antibodies based on their specific application requirements and validated reactivity in their experimental system.

How can Phospho-GRIN1 (Ser896) Antibody be utilized in studying neurological disorders?

This antibody can be instrumental in studying neurological disorders through:

• Comparative analysis of Ser896 phosphorylation levels in post-mortem brain tissue from patients with Alzheimer's disease, schizophrenia, or epilepsy versus controls

• Examining changes in phosphorylation during excitotoxicity models of stroke or traumatic brain injury

• Monitoring alterations in NMDAR phosphorylation in animal models of neuropsychiatric disorders

• Assessing effects of therapeutic compounds on restoring normal NMDAR phosphorylation patterns

• High-throughput screening of drug candidates that modulate PKC-mediated phosphorylation

The phosphorylation status at Ser896 can serve as a biomarker for altered glutamatergic signaling in various pathological conditions where NMDAR dysfunction is implicated .