Phospho-CAMK2B/CAMK2G/CAMK2D (Thr287) Antibody

What is the Phospho-CAMK2B/CAMK2G/CAMK2D (Thr287) Antibody?

The Phospho-CAMK2B/CAMK2G/CAMK2D (Thr287) Antibody is a rabbit polyclonal antibody specifically designed to detect endogenous levels of CaMKII beta, gamma, and delta isoforms, but only when phosphorylated at Threonine 287. This antibody does not recognize non-phosphorylated forms of these proteins, making it a valuable tool for studying the activation state of CaMKII .

The antibody is typically produced by immunizing rabbits with synthetic phosphopeptides corresponding to the amino acid sequence surrounding the Thr287 phosphorylation site (Q-E-T(p)-V-E) derived from human CaMKII beta, gamma, and delta isoforms. Purification is performed using affinity chromatography with epitope-specific phosphopeptides, and non-phospho-specific antibodies are removed through additional chromatographic steps .

What are the technical specifications of this antibody?

The following table summarizes the key technical specifications of the Phospho-CAMK2B/CAMK2G/CAMK2D (Thr287) Antibody:

These specifications are derived from product information provided by commercial suppliers and should be verified with the specific supplier of your antibody.

What are the recommended applications for this antibody?

The Phospho-CAMK2B/CAMK2G/CAMK2D (Thr287) Antibody has been validated for several research applications:

• Western Blot (WB): Most commonly used application, providing specific detection of phosphorylated CaMKII isoforms at 54 and 60 kDa. Optimal conditions include using 30 μg of protein lysate with the antibody at 1 μg/mL concentration .

• Enzyme-Linked Immunosorbent Assay (ELISA): Useful for quantitative measurement of phosphorylated CaMKII in cell or tissue lysates .

• Immunofluorescence (IF): Allows for subcellular localization studies of phosphorylated CaMKII in fixed cells or tissue sections .

• Immunohistochemistry (IHC): Enables detection of phosphorylated CaMKII in tissue sections, particularly useful for studying neuronal distribution patterns .

Each application requires specific optimization for your experimental system, including antibody dilution, incubation conditions, and detection methods.

How should I optimize western blot protocols for detecting phospho-CaMKII?

Based on published protocols, the following optimized western blot procedure is recommended for detecting phospho-CaMKII:

• Sample Preparation: Use 30 μg of whole cell lysate or tissue extract per lane. Include appropriate positive controls such as ionomycin-treated cells (100 nM for 24 hours), which enhances phosphorylation at Thr287 .

• Gel Electrophoresis: Use standard SDS-PAGE to separate proteins, with 8-10% gels typically providing good resolution for CaMKII isoforms.

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane using standard wet or semi-dry transfer systems.

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary Antibody: Incubate with Phospho-CaMKII beta/gamma/delta (Thr287) antibody at 1 μg/mL dilution overnight at 4°C .

• Secondary Antibody: Use an HRP-conjugated anti-rabbit IgG secondary antibody at 0.25 μg/mL (approximately 1:4000 dilution) .

• Detection: Visualize using chemiluminescence. The expected bands for phosphorylated CaMKII beta/gamma/delta appear at approximately 54 and 60 kDa .

• Controls: Include both positive controls (ionomycin-treated samples) and tissue samples known to express CaMKII (e.g., brain tissue from mouse or rat) .

This protocol has been successfully used to detect phosphorylated CaMKII in various cell lines and tissue samples, including SK-N-AS cells, mouse brain, and rat brain .

What controls should be included when using this antibody?

Proper experimental controls are crucial for reliable interpretation of results:

• Positive Controls:

  • Ionomycin-treated cells (100 nM for 24 hours), which enhance phosphorylation at Thr287

  • Brain tissue (mouse or rat), which naturally expresses high levels of CaMKII

  • Recombinant phosphorylated CaMKII proteins (if available)

• Negative Controls:

  • Untreated cell lines (for comparison with stimulated conditions)

  • Samples treated with phosphatase to remove phosphorylation

  • Peptide competition assays, where pre-incubation of the antibody with the immunizing phosphopeptide should block specific signals

• Specificity Controls:

  • When possible, include knockout tissue as the ultimate specificity control (as demonstrated in studies with CaMKII-β knockout mice)

  • Compare results with antibodies from different suppliers or those targeting different epitopes to confirm specificity

Research by Tetenborg et al. (2019) demonstrated the importance of validating antibody specificity using knockout tissues, as they found that some commercial antibodies may cross-react with other proteins, including Cx36 .

How can I differentiate between different CaMKII isoforms in my experiments?

Distinguishing between different CaMKII isoforms requires careful experimental design:

• Molecular Weight Discrimination: CaMKII isoforms have slightly different molecular weights that can sometimes be resolved by SDS-PAGE. CaMKII-α typically runs at 50-54 kDa, CaMKII-β at 60-65 kDa, and CaMKII-γ/δ at similar weights depending on splice variants .

• Isoform-Specific Antibodies: Use antibodies that specifically target individual isoforms in parallel experiments. These can be compared with pan-phospho antibodies to determine which isoforms are phosphorylated .

• Knockout Validation: When available, use tissues or cells from isoform-specific knockout models as controls. For example, CaMKII-β knockout mice provide excellent negative controls for CaMKII-β antibodies .

• Mass Spectrometry: For definitive identification and quantification of specific isoforms and their phosphorylation states, consider using quantitative mass spectrometry approaches, which can distinguish between closely related proteins and identify multiple phosphorylation sites simultaneously .

• Subcellular Fractionation: Different isoforms and their phosphorylated forms show distinct subcellular localization patterns. For example, Thr287-phosphorylated CaMKII-β is enriched in synaptic fractions, while other phosphorylated forms may be more abundant in cytosolic fractions .

What is the functional significance of Thr287 phosphorylation in CaMKII?

Phosphorylation at Thr287 (in CaMKII-β/γ/δ) or the homologous Thr286 (in CaMKII-α) represents a critical regulatory event with profound functional implications:

• Autonomous Activity: Thr287 phosphorylation enables CaMKII to maintain its activity even after calcium levels decrease, effectively serving as a molecular memory mechanism. This autonomous activity is essential for various forms of synaptic plasticity, including long-term potentiation (LTP) .

• Synaptic Localization: Phosphorylation at Thr287 alters the subcellular distribution of CaMKII, with phosphorylated forms showing enrichment in synaptic fractions compared to cytosolic compartments . This translocation to synapses is crucial for modifying synaptic strength.

• Protein Interaction Networks: Thr287-phosphorylated CaMKII interacts with a distinct set of proteins compared to non-phosphorylated forms, including components of the postsynaptic density (PSD) and glutamatergic synapses .

• Signal Amplification: The autophosphorylation mechanism at Thr287 serves as a signal amplification step, translating transient calcium signals into sustained kinase activity, which is critical for converting short-term stimuli into long-term cellular changes .

• Cross-talk with Other Phosphorylation Sites: Thr287 phosphorylation influences the phosphorylation state of other sites within CaMKII. For example, the T286A mutation in CaMKII-α significantly reduces phosphorylation at Ser275 and affects phosphorylation of CaMKII-β at Ser315 and Thr320/Thr321 .

Understanding the precise functional consequences of Thr287 phosphorylation remains an active area of research, with important implications for neurological function and disease.

How do phosphorylation patterns differ across subcellular compartments?

Research using subcellular fractionation and phospho-specific antibodies has revealed distinct patterns of CaMKII phosphorylation across different cellular compartments:

• Synaptic Enrichment: Thr286-phosphorylated CaMKII-α and Thr287-phosphorylated CaMKII-β show 5-fold and 2.4-fold higher levels in synaptic (Triton-insoluble) fractions compared to cytosolic fractions, respectively. This suggests specific targeting of active kinase to synaptic structures .

• Cytosolic Phosphorylation Patterns: In contrast, other phosphorylation sites show preferential enrichment in cytosolic fractions. For example, Thr306-phosphorylated CaMKII-α and Ser315/Thr320/Thr321-phosphorylated CaMKII-β are selectively enriched in cytosolic fractions rather than synaptic compartments .

• Membrane Association: Intermediate levels of Thr286/Thr287 phosphorylation are typically found in membrane (Triton-soluble) fractions, suggesting a transition state or separate regulatory mechanism in these compartments .

• Activity-Dependent Redistribution: Neuronal activation can dynamically alter these distribution patterns, with stimuli that increase intracellular calcium promoting translocation of phosphorylated CaMKII to synaptic sites .

• Cell-Type Specific Patterns: Within the retina, CaMKII-β shows specific localization patterns in bipolar cell terminals, where it may regulate both gap junctions and ribbon synapses .

These differential phosphorylation patterns likely reflect specialized functions of CaMKII in different cellular compartments and provide important insights into the spatial regulation of signaling networks.

What are the known interactions between CaMKII phosphorylation and other signaling pathways?

CaMKII phosphorylation is integrated with numerous other signaling pathways, creating a complex regulatory network:

• Glutamatergic Signaling: Phosphorylated CaMKII interacts with components of glutamatergic synapses, including NMDA receptors, AMPA receptors, and various scaffold proteins. Gene ontology analysis of CaMKII-associated phosphoproteins shows significant enrichment for terms such as "postsynaptic specialization," "glutamatergic synapse," and "postsynaptic organization" .

• Gap Junction Regulation: CaMKII-β may regulate Cx36-containing gap junctions in bipolar cell terminals of the retina, suggesting a role in electrical coupling between neurons .

• Cytoskeletal Regulation: Many CaMKII substrates are involved in cytoskeletal organization, indicating a role in structural plasticity of dendritic spines and synapses .

• Crosstalk with Phosphatases: CaMKII-mediated phosphorylation is counterbalanced by various phosphatases, creating dynamic regulation of substrate phosphorylation states.

• Isoform-Specific Functions and Compensation: Studies using knockout models suggest that while CAMK2A and CAMK2B can have distinct roles, they can also partially compensate for each other in certain brain functions .

• Integration with Other Calcium-Dependent Pathways: CaMKII phosphorylation can be modulated by other calcium-sensing proteins and pathways, creating complex feedback and feedforward regulatory mechanisms.

What are common issues when using phospho-specific antibodies and how can they be resolved?

Researchers commonly encounter several challenges when working with phospho-specific antibodies, including:

• Low Signal Strength:

  • Potential Cause: Rapid dephosphorylation during sample preparation.

  • Solution: Include phosphatase inhibitors in lysis buffers and maintain samples at cold temperatures throughout processing. Consider short-term treatments with phosphatase inhibitors such as okadaic acid or calyculin A before harvesting cells.

• High Background or Non-specific Bands:

  • Potential Cause: Cross-reactivity with similar phosphorylation motifs or non-phosphorylated proteins.

  • Solution: Optimize blocking conditions (try both BSA and milk), increase washing steps, reduce antibody concentration, and consider using phospho-peptide blocking controls to identify specific signals .

• Variable Results Between Experiments:

  • Potential Cause: Phosphorylation states can change rapidly due to stress or handling.

  • Solution: Standardize sample collection procedures, minimize time between tissue collection and processing, and include positive controls in each experiment.

• Discrepancies Between Different Antibodies:

  • Potential Cause: Different antibodies may recognize slightly different epitopes or conformations.

  • Solution: Validate findings with multiple antibodies when possible and confirm specificity using knockout tissues or cells when available .

• Loss of Signal During Storage:

  • Potential Cause: Degradation of phosphorylation over time.

  • Solution: Prepare fresh samples when possible, or add additional phosphatase inhibitors for storage. Consider storing samples in SDS-PAGE loading buffer at -80°C.

How can I interpret western blot data for phospho-CaMKII across different experimental conditions?

Proper interpretation of phospho-CaMKII western blot data requires careful consideration of several factors:

• Normalization Strategy:

  • Total Protein Loading: Use total protein stains (e.g., Ponceau S) to normalize for loading variations.

  • Total CaMKII: Probe parallel blots or strip and reprobe for total CaMKII to calculate the phospho-to-total ratio, which indicates the proportion of activated kinase.

  • Housekeeping Proteins: While commonly used, these may vary across experimental conditions and should be validated.

• Multiple Band Interpretation:

  • The 54 and 60 kDa bands observed in western blots correspond to different CaMKII isoforms .

  • Additional bands may represent splice variants, degradation products, or cross-reactivity.

  • Compare band patterns with isoform-specific antibodies and knockout controls when available .

• Quantification Across Conditions:

  • Establish a linear detection range for your imaging system.

  • Use biological replicates (n≥3) for statistical analysis.

  • Consider the relative change in phosphorylation rather than absolute values, which can vary between experiments.

• Temporal Dynamics:

  • CaMKII phosphorylation is dynamic, with rapid changes in response to stimuli.

  • Consider time-course experiments to capture the full response profile.

  • Ionomycin treatment (100 nM for 24 hours) can serve as a positive control for increased Thr287 phosphorylation .

• Subcellular Fractionation:

  • Different phosphorylation patterns exist across subcellular compartments .

  • Consider analyzing cytosolic, membrane, and synaptic fractions separately for a more complete picture.

What are emerging applications for studying CaMKII phosphorylation in neurological disorders?

CaMKII phosphorylation has been implicated in various neurological disorders, opening new research avenues:

• Neurodegenerative Diseases:

  • Altered CaMKII phosphorylation patterns have been observed in Alzheimer's disease models, suggesting potential roles in synaptic dysfunction and neurodegeneration.

  • Targeting CaMKII pathways may offer therapeutic possibilities for protecting synaptic function.

• Epilepsy Research:

  • CaMKII activity is dysregulated in various epilepsy models, and modulating its phosphorylation may affect seizure susceptibility and progression.

  • Phospho-specific antibodies enable monitoring of CaMKII activation states in epileptic tissues.

• Learning and Memory Disorders:

  • As a key molecular substrate for synaptic plasticity, CaMKII phosphorylation is directly relevant to cognitive disorders.

  • Studies using phospho-CaMKII antibodies can provide mechanistic insights into cognitive impairments and potential therapeutic targets.

• Psychiatric Disorders:

  • Emerging evidence links CaMKII signaling abnormalities to various psychiatric conditions, including schizophrenia and depression.

  • Monitoring phosphorylation states across different brain regions may provide biomarkers for these conditions.

• Stroke and Ischemia:

  • CaMKII phosphorylation changes dramatically following ischemic events, potentially contributing to excitotoxicity.

  • Therapeutic strategies targeting these phosphorylation events are being explored.

Future research will likely focus on developing more specific modulators of CaMKII phosphorylation for potential therapeutic applications and more sensitive detection methods for diagnostic purposes.

How can phospho-CaMKII antibodies be used in combination with other techniques for comprehensive analysis?

Integrating phospho-CaMKII antibody-based methods with complementary techniques enhances research depth:

• Mass Spectrometry Integration:

  • Immunoprecipitation with phospho-CaMKII antibodies followed by mass spectrometry can identify interacting partners specific to the phosphorylated state.

  • Quantitative phosphoproteomics can map the full spectrum of CaMKII substrates and their phosphorylation dynamics .

• Super-Resolution Microscopy:

  • Combining phospho-specific antibodies with techniques like STORM or STED microscopy enables nanoscale visualization of phosphorylated CaMKII distribution within synapses.

  • This approach has revealed distinct nanodomain organization of activated CaMKII.

• Electrophysiology Correlation:

  • Parallel analysis of CaMKII phosphorylation states and electrophysiological recordings can link molecular changes to functional outcomes.

  • This is particularly valuable for understanding synaptic plasticity mechanisms.

• In Vivo Imaging:

  • Development of phosphorylation-sensitive fluorescent reporters enables real-time monitoring of CaMKII activation in living neurons.

  • These can be combined with behavioral assays to correlate molecular events with learning and memory.

• Single-Cell Analysis:

  • Combining phospho-CaMKII immunostaining with single-cell RNA-seq or spatial transcriptomics provides insights into cell-type-specific phosphorylation patterns and their relationship to gene expression profiles.

• Computational Modeling:

  • Data from phospho-CaMKII studies can inform computational models of synaptic plasticity and neuronal network function.

  • These models can generate testable predictions about CaMKII regulation and function.

These integrated approaches promise to provide a more comprehensive understanding of CaMKII phosphorylation in neuronal function and pathology.