CAMK2N1 Antibody, Biotin conjugated

What is CAMK2N1 and what are its primary biological functions?

CAMK2N1 is a small 78 amino acid (9 kDa calculated, 10 kDa observed) protein that functions as an endogenous inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII) . Research demonstrates that CAMK2N1 plays critical roles in multiple physiological systems:

• Cardiovascular regulation: Acts as a negative regulator of blood pressure and left ventricular mass

• Metabolic function: Influences insulin sensitivity and adipose tissue regulation

• Cancer biology: Functions as a potential tumor suppressor gene, with expression significantly downregulated in prostate cancer tissues compared to normal tissues

CAMK2N1 exerts these effects by modulating CaMKII activity, a central kinase involved in calcium signaling pathways across multiple cell types.

What applications are most suitable for biotin-conjugated CAMK2N1 antibodies?

Biotin-conjugated CAMK2N1 antibodies are particularly valuable for:

• Immunohistochemistry (IHC): Validated for tissue section analysis with recommended dilutions of 1:200-1:800

• ELISA: For quantitative detection of CAMK2N1 in research samples

• Immunoprecipitation: Biotin conjugation enables streptavidin-based pulldown of protein complexes

• Multi-color immunofluorescence: Allows for combination with other antibodies using streptavidin detection systems

• Chromatin immunoprecipitation: For studying CAMK2N1 interactions with genomic regions

Methodological Consideration: When using biotin-conjugated antibodies, it's essential to block endogenous biotin in tissue samples using streptavidin/biotin blocking kits, particularly in tissues with high endogenous biotin (brain, kidney, liver).

What tissues express CAMK2N1 and what is its subcellular localization?

CAMK2N1 shows distinctive expression patterns that researchers should consider when designing experiments:

Subcellular localization: While primarily cytoplasmic, CAMK2N1 can translocate to interact with CaMKII at specific subcellular compartments depending on cellular context.

How should researchers validate the specificity of biotin-conjugated CAMK2N1 antibodies?

Proper validation is critical for antibody-based research. For CAMK2N1 antibodies, consider these approaches:

• Positive and negative tissue controls: Use tissues known to express or lack CAMK2N1 (brain tissue serves as an excellent positive control)

• Peptide competition assay: Pre-incubate antibody with immunogen peptide to confirm specificity

• Knockout/knockdown validation: Test antibody in CAMK2N1 knockout or knockdown samples

• Western blot confirmation: Verify single band at expected molecular weight (10 kDa)

• Cross-reactivity testing: Test against related CaMKII inhibitor proteins

Critical consideration: When interpreting results, note that CAMK2N1 expression can be significantly altered in disease states, with studies showing downregulation in prostate cancer (correlation coefficient with PRMT5: r = -0.4279, P < 0.01) .

What are the optimal fixation and antigen retrieval methods for CAMK2N1 immunohistochemistry?

Based on experimental evidence, the following protocol optimizes CAMK2N1 detection:

Technical note: For mouse brain tissue, the suggested antigen retrieval with TE buffer pH 9.0 has been empirically validated to produce optimal results .

How does CAMK2N1 interact with and regulate CaMKII activity at the molecular level?

CAMK2N1 exhibits sophisticated regulation of CaMKII through multiple mechanisms:

• Direct binding interaction: CAMK2N1 binds to specific regions of CaMKII, particularly targeting:

  • The ATP-binding region (amino acids 19-68)

  • S-site (amino acids 130-164)

  • T-site regions (amino acids 241-269)

  • The autoregulatory domain (amino acids 280-317)

• Inhibitory mechanism: CAMK2N1 contains regions homologous to natural CaMKII inhibitor peptides, with studies showing that the N-terminal region (AKAP18δ-N) inhibits CaMKIIδ through binding to regions homologous to natural CaMKII inhibitor peptide

• Calmodulin competition: CAMK2N1 may also modulate CaMKII activity through interaction with calmodulin, introducing a second level of control

Research applications examining these interactions often require combining antibody-based detection with protein interaction studies such as co-immunoprecipitation or proximity ligation assays.

What are the implications of CAMK2N1 in cardiovascular and metabolic disease research?

CAMK2N1 has emerged as a significant regulatory factor in cardiometabolic disease, with knockout studies in rats revealing:

• Cardiovascular effects:

  • Reduced mean systolic BP (-Δ12 mm Hg, P<0.001) and diastolic BP (-Δ10 mm Hg, P<0.005) in CAMK2N1−/− rats compared to SHR controls

  • 50% reduction in CaMKII activity

  • Increased renal ACE2 activity and Ang(1-7) concentrations

  • Elevated renal and serum eNOS and serum nitrate levels

• Metabolic effects:

  • Reduced insulin resistance

  • Decreased visceral fat and adipogenic capacity

  • Alterations in cell cycle and complement pathways, independent of CaMKII

• Human correlation:

  • CAMK2N1 expression in visceral fat correlates with adiposity

  • Genomic variants increasing CAMK2N1 expression associate with increased risk of coronary artery disease and type 2 diabetes mellitus

When designing experiments to investigate these pathways, researchers should consider combining CAMK2N1 detection with functional readouts of CaMKII activity and downstream physiological parameters.

How can researchers effectively analyze CAMK2N1-mediated CaMKII regulation in different experimental systems?

To comprehensively analyze CAMK2N1-CaMKII interactions across experimental systems:

• Biochemical analysis:

  • CaMKII activity assays using synthetic peptide substrates

  • Phosphorylation assessment of known CaMKII targets (e.g., PLN, RYR)

  • Co-immunoprecipitation studies using biotin-conjugated CAMK2N1 antibodies

• Cellular models:

  • Fluorescence resonance energy transfer (FRET) to detect CaMKII-CAMK2N1 interactions

  • Immunofluorescence to track subcellular localization changes

  • Live-cell calcium imaging to correlate with CaMKII activity

• Tissue/in vivo analysis:

  • Immunohistochemistry to map expression patterns (dilution 1:200-1:800)

  • Tissue-specific knockout models

  • Physiological parameter measurements (e.g., blood pressure, metabolic indices)

Important consideration: Research has demonstrated that CAMK2N1 may not function exclusively as a CaMKII inhibitor in all contexts. In fact, knockout studies suggest "that endogenous Camk2n1/CAMK2N1 may not function exclusively as an inhibitor of CaMKII and requires a reappraisal of existing studies that have used nonspecific CaMKII inhibitors proposed to mimic Camk2n1 function" .

What are common challenges when using biotin-conjugated CAMK2N1 antibodies and how can they be overcome?

Research insight: When working with brain tissue, where CaMKII is highly abundant, optimization of blocking conditions is particularly important to distinguish specific CAMK2N1 signal from background.

How should researchers interpret changes in CAMK2N1 expression in disease models?

Interpretation of CAMK2N1 expression changes requires careful consideration of:

• Context-dependent function:

  • In cancer: CAMK2N1 downregulation correlates with tumor progression (r = -0.4279 correlation with PRMT5 in prostate cancer)

  • In cardiovascular disease: CAMK2N1 expression positively correlates with elevated blood pressure and left ventricular mass

  • In metabolic disease: Expression in visceral fat correlates with adiposity

• Regulatory mechanisms:

  • Epigenetic regulation: PRMT5 can catalyze histone methylation at the CAMK2N1 promoter

  • Genetic variants: cis-eQTLs regulating CAMK2N1 expression associate with cardiometabolic traits

• Functional consequences:

  • CaMKII-dependent effects: Changes in phosphorylation of CaMKII targets

  • CaMKII-independent effects: Altered cell cycle and complement pathways

Research perspective: When publishing CAMK2N1 expression data, include both protein and mRNA measurements, along with functional readouts of CaMKII activity to provide comprehensive mechanistic insight.

How do experimental conditions affect CAMK2N1-CaMKII interactions and what controls should be included?

The CAMK2N1-CaMKII interaction is highly sensitive to experimental conditions:

• Calcium concentration:

  • Low calcium: Minimal CaMKII activation, CAMK2N1 binding may be reduced

  • High calcium: CaMKII activation and autophosphorylation may alter CAMK2N1 binding kinetics

• Phosphorylation status:

  • CaMKII autophosphorylation at Thr286 allows it to remain active even after calcium signals return to baseline

  • This modification may affect CAMK2N1 binding affinity

• Essential controls:

  • Calcium-free conditions

  • Calcium/calmodulin supplementation

  • CaMKII inhibitor controls (e.g., KN-93, which has been used at 10 μM concentration in published studies)

  • Phosphorylation state-specific antibodies for CaMKII

Research recommendation: When investigating CAMK2N1-CaMKII interactions, researchers should conduct experiments under both basal and stimulated conditions to capture the dynamic nature of this regulatory system.

How might biotin-conjugated CAMK2N1 antibodies be used to study novel signaling interactions?

Emerging research areas where biotin-conjugated CAMK2N1 antibodies could provide valuable insights:

• Proximity-dependent biotinylation (BioID/TurboID):

  • Fusion of biotin ligase to CAMK2N1 to identify proximal interacting proteins

  • Verification of interactions using biotin-conjugated antibodies

• Single-cell analysis:

  • Combination with other markers for multi-parameter flow cytometry

  • Single-cell western blotting techniques for heterogeneous populations

• Spatial transcriptomics correlation:

  • Combined protein (using biotin-conjugated antibodies) and RNA detection

  • Correlation of CAMK2N1 protein localization with gene expression patterns

• In vivo tracking:

  • Intravital microscopy using streptavidin-fluorophore detection

  • Whole-animal imaging of CAMK2N1 distribution in disease models

Research frontier: Recent studies suggest CAMK2N1 may have CaMKII-independent functions, opening new avenues for research beyond the traditional focus on CaMKII inhibition .

What are the implications of CAMK2N1 in therapeutic target development and how can antibodies facilitate this research?

CAMK2N1 shows significant potential as a therapeutic target, with antibody-based research facilitating development:

• Target validation:

  • Biotin-conjugated antibodies enable precise localization in disease tissues

  • Correlation of expression with disease progression (e.g., CAMK2N1 downregulation in prostate cancer)

• Mechanism exploration:

  • Investigation of CAMK2N1-CaMKII binding interfaces using epitope-specific antibodies

  • Structure-function studies to guide small molecule development

• Therapeutic screening:

  • Antibody-based assays to screen compounds that modulate CAMK2N1 expression or function

  • Monitoring CAMK2N1 levels in response to experimental therapeutics

Clinical relevance: Research suggests that "therapeutic targeting of CAMK2N1 may allow amelioration of MetS features in humans" , highlighting the translational significance of basic research in this area.