CAMK2D Antibody

What is CAMK2D and why is it an important research target?

CAMK2D (calcium/calmodulin-dependent protein kinase II delta) is a member of the CAMK Ser/Thr protein kinase family that plays crucial roles in cellular signaling. In humans, the canonical CAMK2D protein has 499 amino acid residues with a molecular mass of 56.4 kDa and localizes primarily to the cell membrane . It is predominantly expressed in cardiac and skeletal muscle tissues and participates in peptidyl-serine phosphorylation processes . Recent research has implicated CAMK2D in various physiological and pathological conditions, including cardiac function, renal physiology, and certain cancers like malignant mesothelioma, making it a significant target for ongoing research .

What applications are most common for CAMK2D antibodies?

CAMK2D antibodies are widely employed in several experimental techniques:

These applications have generated over 470 citations in scientific literature, demonstrating the robustness and reliability of CAMK2D antibodies in research settings .

How do researchers address potential cross-reactivity with other CAMK2 isoforms?

Due to high sequence homology among CAMK2 family members, antibodies raised against CAMK2D may cross-react with CAMK2A, CAMK2B, or CAMK2G . To address this challenge:

• Validate antibody specificity using knockout or knockdown controls

• Choose antibodies targeting unique regions of CAMK2D

• Employ isoform-specific antibodies that recognize distinctive epitopes

• Perform parallel experiments with multiple antibodies recognizing different epitopes

• Include appropriate negative controls in experimental designs

Many commercial CAMK2D antibodies specify potential cross-reactivity in their documentation, allowing researchers to select the most appropriate reagent for their experimental needs .

How can phospho-specific CAMK2D antibodies be utilized to study its activation state?

CAMK2D undergoes autophosphorylation at multiple sites that regulate its activity and substrate specificity. When designing experiments with phospho-specific antibodies:

• Experimental Design Strategy: Use site-specific phospho-antibodies in combination with pan-CAMK2D antibodies to simultaneously monitor total protein levels and phosphorylation state .

• Stimulus-Response Monitoring: Activate CAMK2D with calcium/calmodulin or other physiological stimuli, then detect temporal phosphorylation changes using Western blot or immunofluorescence with phospho-specific antibodies .

• Quantification Method: When quantifying phosphorylation levels in immunoblotting, always normalize phospho-CAMK2D signal to total CAMK2D protein to account for expression level variations between samples .

• Validation Approach: Confirm antibody specificity using phosphatase treatment, phosphomimetic mutants, or CRISPR-edited cell lines with phospho-site mutations .

Recent phosphoproteomic studies following CRISPR-Cas9-mediated CAMK2D knockout have identified 169 significantly decreased and 206 increased phosphosites among 11,570 quantified sites, providing valuable insights for researchers studying CAMK2D-dependent phosphorylation events .

What are optimal strategies for detecting CAMK2D in tissue samples with heterogeneous expression?

When working with tissue samples where CAMK2D expression varies by cell type:

• Sample Preparation Protocol: For IHC in brain tissue, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may be used alternatively . This optimization is crucial as inadequate antigen retrieval can produce false negative results.

• Signal Amplification Approach: For tissues with low expression, employ tyramide signal amplification or highly sensitive detection systems to enhance signal while maintaining specificity .

• Co-localization Analysis: Perform dual immunostaining with cell-type specific markers (e.g., GFAP for astrocytes in brain samples) to definitively identify CAMK2D-expressing cells .

• Sequential Sectioning Technique: Use serial section analysis comparing CAMK2D staining with known expression patterns of other markers to confirm specificity .

An exemplary application of this approach revealed CAMK2D protein expression in GFAP+ lateral ventricle quiescent neural stem cells (LV qNSCs) surrounding subep-ChAT+ neurons, providing important insights into neural development mechanisms .

How can CAMK2D antibodies be integrated with CRISPR-Cas9 technology for functional studies?

The integration of CAMK2D antibodies with CRISPR-Cas9 techniques provides powerful tools for functional analysis:

• Knockout Validation Strategy: When generating CAMK2D knockout cell lines, antibodies provide essential validation of knockout efficiency through Western blotting, as demonstrated in studies where guide RNAs targeting exons 8 and 9 (coding for the kinase domain) successfully ablated CAMK2D expression .

• Phenotypic Analysis Framework: After CAMK2D deletion, employ antibodies in immunoblotting, immunofluorescence, and phosphoproteomic analyses to comprehensively characterize cellular changes, as shown in studies of collecting duct cells where knockout impacted AQP2 protein abundance .

• Substrate Identification Workflow: Combine CAMK2D antibodies with phosphoproteomic analysis of knockout vs. wild-type cells to identify downstream targets, revealing both direct and indirect phosphorylation events regulated by CAMK2D .

• Structure-Function Analysis: Use CAMK2D antibodies to validate expression of various CAMK2D mutants in complementation experiments, allowing for detailed investigation of specific domains or phosphorylation sites .

This approach was successfully employed to identify that CAMK2D knockout in mpkCCD cells reduced AQP2 protein abundance and decreased AQP2 phosphorylation at Ser256 and Ser269, while still maintaining AQP2 trafficking to and from the apical plasma membrane .

How should researchers address discrepancies in observed molecular weight of CAMK2D in Western blot experiments?

Researchers frequently encounter variations in the observed molecular weight of CAMK2D:

• Expected vs. Observed Weight Analysis: While the calculated molecular weight of CAMK2D is approximately 59 kDa, observed weights in Western blots may range from 54-59 kDa or approximately 64 kDa , depending on experimental conditions and isoform detection.

• Isoform Identification Protocol: To definitively identify specific isoforms:

  • Use isoform-specific antibodies when available

  • Compare migration patterns with recombinant protein standards

  • Employ knockout/knockdown controls for each isoform

  • Consider RT-PCR to identify expressed isoform transcripts in your sample

• Post-translational Modification Assessment: CAMK2D undergoes various post-translational modifications that affect migration. Treatments with phosphatases, glycosidases, or other enzymes prior to SDS-PAGE can help determine modification status .

• Sample Preparation Optimization: Different lysis buffers and denaturation conditions can affect observed molecular weight. Standardize these parameters across experiments and include positive controls with known CAMK2D expression .

What strategies can improve specificity when studying CAMK2D in BAP1-deficient malignant mesothelioma?

Recent research has identified CAMK2D as a promising diagnostic and therapeutic target for BAP1-deficient malignant mesothelioma (MMe) . When investigating this relationship:

• Antibody Selection Guidelines: Choose antibodies validated in mesothelioma tissue or cell lines. Polyclonal antibodies purified by antigen-affinity chromatography offer good specificity for this application .

• Correlation Analysis Framework: When examining the relationship between BAP1 and CAMK2D expression:

  • Use serial sections for IHC analysis

  • Employ dual immunofluorescence staining

  • Quantify expression levels using standardized scoring systems

• Experimental Controls Implementation: Include both BAP1-positive and BAP1-negative mesothelioma samples as controls, as studies have shown that CAMK2D is highly expressed in 70% of human MMe tissues (56/80) and correlates with loss of BAP1 expression .

• Expression Pattern Assessment: Based on immunohistochemical analysis of 80 MMe tissue samples, expect to observe strong (3+), moderate (2+), or weak (1+) CAMK2D-positive signals. Note that the positivity rate for CAMK2D in BAP1-negative MMe tissues (94%) is significantly higher than in BAP1-positive MMe tissues (33%) .

What are the optimal storage and handling conditions for maintaining CAMK2D antibody activity?

To ensure reliable and reproducible results when working with CAMK2D antibodies:

• Storage Temperature Requirements: Most CAMK2D antibodies should be stored at -20°C and remain stable for one year after shipment . For reconstituted antibodies, storage at -20°C to -70°C under sterile conditions is recommended for up to 6 months .

• Buffer Composition Guidelines: Typical storage buffers contain PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Some smaller antibody aliquots (20μl sizes) may contain 0.1% BSA as a stabilizer .

• Aliquoting Recommendations: While aliquoting is generally unnecessary for -20°C storage , for frequently used antibodies, dividing into single-use aliquots minimizes freeze-thaw cycles that can reduce activity.

• Reconstitution Protocol: For lyophilized antibodies, reconstitute to the recommended concentration using sterile buffer. After reconstitution, these can typically be stored at 2-8°C under sterile conditions for approximately 1 month .

• Freeze-Thaw Cycle Limitation: To maintain antibody performance, use a manual defrost freezer and avoid repeated freeze-thaw cycles , as this can lead to reduced binding efficiency and increased background signal.

How might single-cell techniques be integrated with CAMK2D antibody detection methods?

As single-cell analysis technologies advance, several approaches show promise for CAMK2D research:

• Single-Cell Western Blot Application: Emerging microfluidic platforms allow Western blot analysis at the single-cell level, potentially revealing heterogeneity in CAMK2D expression and phosphorylation states within tissues that would be masked in bulk analysis .

• CyTOF Integration Strategy: Mass cytometry using CAMK2D antibodies conjugated to metal isotopes can enable simultaneous detection of multiple markers alongside CAMK2D, providing rich contextual data about CAMK2D-expressing cells .

• Spatial Transcriptomics Correlation: Combining immunofluorescence detection of CAMK2D protein with spatial transcriptomics can reveal relationships between CAMK2D protein levels and mRNA expression of related pathway components at cellular resolution .

• Live-Cell Imaging Adaptation: Developing non-disruptive labeling techniques using CAMK2D antibody fragments or nanobodies could enable tracking of CAMK2D dynamics in living cells over time .

These emerging techniques will likely provide unprecedented insights into the heterogeneity of CAMK2D expression, localization, and function across different cell types within complex tissues.

What role can CAMK2D antibodies play in understanding neurodevelopmental disorders?

Recent research has begun to uncover important roles for CAMK2D in neurodevelopment , suggesting several promising research directions:

• Variant Analysis Framework: CAMK2D antibodies can be used to study the impact of genetic variants (e.g., R275H, L291F) on protein expression, stability, and localization in cellular models of neurodevelopmental disorders .

• Developmental Expression Mapping: Tracking CAMK2D expression throughout neural development using antibody-based techniques can identify critical periods where CAMK2D function may impact neurodevelopment .

• Circuit-Level Investigation Approach: As suggested by recent findings on cholinergic signaling pathways, CAMK2D antibodies can help explore how neural circuit activity modulates CAMK2D signaling in neural stem cells, potentially influencing brain development .

• Therapeutic Target Validation: For neurological conditions associated with CAMK2D dysfunction, antibodies provide essential tools for validating target engagement of potential therapeutic compounds in both in vitro and in vivo models .

This research direction is particularly relevant given recent findings showing CAMK2D protein colocalization with M3 receptors on lateral ventricle quiescent neural stem cells within the ventral subventricular zone, suggesting a role in neural stem cell regulation .