CAMK2N2 Antibody

What is CAMK2N2 and what cellular functions does it regulate?

CAMK2N2 (Calcium/calmodulin-dependent protein kinase II inhibitor 2) is a potent and specific endogenous inhibitor of CaMKII that acts by trapping Ca²⁺/calmodulin on CAMK2 . It has a molecular weight of approximately 8.7 kDa in humans with 79 amino acid residues and is localized in both the nucleus and cytoplasm .

CAMK2N2 plays important roles in:

• Regulation of calcium/calmodulin-dependent protein kinase II activity

• Potential modulation of cell growth and proliferation

• Neuronal signaling pathways

Studies have shown that CAMK2N2 may play a regulatory role in cell growth when overexpressed in colon adenocarcinoma LoVo cells . Additionally, its interaction with CAMK2 affects numerous downstream processes since CAMK2 phosphorylates nearly 40 different proteins including enzymes, ion channels, kinases, and transcription factors .

How do CAMK2N2 isoforms differ in tissue distribution and function?

CAMK2N2 exists in two main isoforms with distinct tissue distribution patterns:

Both isoforms inhibit CaMKII, but their differential expression suggests tissue-specific regulatory roles. In cultured mature hippocampal neurons, CaM-KIIN is present in cell bodies and dendrites but, unlike CaMKII, does not display punctate staining at synapses .

What are the optimal applications for CAMK2N2 antibodies in neuroscience research?

CAMK2N2 antibodies are valuable tools for investigating CaMKII regulation in neuronal systems. Based on validation data, the following applications show reliable results:

When studying CAMK2N2 in brain tissue, researchers should note that immunohistochemical analysis shows stronger expression in specific brain regions (frontal cortex, hippocampus for CaM-KIINalpha; cerebellum and hindbrain for CaM-KIINbeta) .

How can I validate CAMK2N2 antibody specificity for my experimental model?

Proper validation of CAMK2N2 antibody specificity is crucial for reliable research outcomes:

• Cross-reactivity assessment: Verify antibody specificity using tissues from multiple species (human, mouse, rat) as CAMK2N2 shares high sequence homology across species (e.g., mouse and rat orthologs show 96% identity with human) .

• Knockout/knockdown controls: Use CAMK2N2 knockout tissue or knockdown cells as negative controls. For knockdown, validated shRNA sequences targeting mouse Camk2d include:

  • 5′-GCAACTGATTGAAGCTATCAA-3′

  • 5′-GCATAGACTGTATCAGCAGAT-3′

  • 5′-CCTGAAGCATTGGGCAACTTA-3′

• Peptide competition assay: Pre-incubate the antibody with immunizing peptide (e.g., "ILPYSEDKMGR FGADPEGSDL SFSCR" for some commercial antibodies) to confirm signal specificity.

• Multiple antibody validation: Compare results using antibodies raised against different epitopes of CAMK2N2 to confirm consistent patterns.

How can I effectively design experiments to study CAMK2N2-CAMK2 interactions?

To investigate CAMK2N2-CAMK2 interactions, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-CAMK2N2 antibodies to pull down protein complexes

  • Verify interaction by immunoblotting for CAMK2 subunits (α, β, γ, δ)

  • Include proper controls (IgG, lysate input)

• Yeast two-hybrid screening:

  • This method was successfully used to identify CaM-KIINalpha

  • Use CAMK2 as bait to identify novel interacting proteins

• Functional inhibition assays:

  • Compare CaMKII activity in the presence/absence of CAMK2N2

  • Measure phosphorylation of known CAMK2 substrates

• Overexpression and knockdown studies:

  • Use validated expression constructs for CAMK2N2

  • For knockdown experiments, use the shRNA sequences mentioned in section 2.2

  • Assess changes in CAMK2 activity and downstream signaling

What are the key considerations when using CAMK2N2 antibodies for brain tissue analysis?

When analyzing CAMK2N2 in brain tissue:

• Fixation protocol optimization:

  • Immersion-fixed paraffin-embedded sections yield good results

  • For IHC-F (frozen sections), antibody dilutions of 1:100-500 are recommended

• Antigen retrieval:

  • Heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) improves signal quality

  • Alternative methods include citrate buffer (pH 6.0)

• Region-specific considerations:

  • Account for differential expression of CAMK2N2 isoforms across brain regions

  • CaM-KIINalpha shows stronger expression in frontal cortex and hippocampus

  • CaM-KIINbeta shows stronger expression in cerebellum and hindbrain

• Counter-staining strategies:

  • Hematoxylin counter-staining can help identify cellular structures

  • Consider co-staining with neuronal markers for more detailed analysis

How does FUS-ΔNLS mutation affect CAMK2N2 expression in ALS models?

Research has demonstrated a significant relationship between FUS-ΔNLS mutation (associated with amyotrophic lateral sclerosis) and CAMK2N2 expression:

• Transcriptional regulation: FUS-ΔNLS increases CAMK2N2 at both mRNA and protein levels, whereas wild-type FUS downregulates CAMK2N2 mRNA .

• Quantitative effects:

  • FUS-ΔNLS causes approximately 2-fold increase in CAMK2N2 mRNA

  • Protein levels increase by approximately 3.5-fold

  • These changes are observed in both stable and transient transfections

• Mechanism: FUS-ΔNLS accumulates on the CAMK2N2 promoter (3.5-fold increase compared to WT-FUS binding at 48 hours), suggesting direct transcriptional regulation .

• Temporal dynamics: The effect progresses over time with steady increases observed at 6, 24, and 48 hours after FUS expression .

These findings identify CAMK2N2 as the first direct target of FUS-ΔNLS, suggesting potential pathological mechanisms in ALS involving calcium signaling disruption.

What role does CAMK2N2 play in sleep regulation and how can antibodies help investigate this function?

Recent research suggests connections between CAMK2 and sleep regulation, with potential implications for CAMK2N2:

• CAMK2 subunit involvement:

  • CaMKIIα knockout mice show reduced basal sleep and altered sleep homeostasis

  • CaMKIIβ knockout mice maintain normal basal sleep but show reduced sleep rebound after deprivation

• Research approach using antibodies:

  • Use CAMK2N2 antibodies to map expression patterns in sleep-regulating brain regions

  • Compare CAMK2N2 levels in sleep-deprived versus normal conditions

  • Investigate co-localization with CaMKIIα and CaMKIIβ in relevant neuronal populations

• Functional studies:

  • Combine CAMK2N2 antibody-based detection with electrophysiological recordings

  • Correlate CAMK2N2 expression with sleep EEG parameters

  • Use pharmacological or genetic manipulation of CAMK2N2 to assess sleep outcomes

Understanding CAMK2N2's role in sleep regulation could provide insights into sleep disorders and potential therapeutic targets.

How can I use CAMK2N2 antibodies to investigate cancer cell signaling pathways?

CAMK2N2 has been implicated in cancer biology, particularly in cell growth regulation. Researchers can use CAMK2N2 antibodies to:

• Expression profiling across cancer types:

  • Compare CAMK2N2 levels between normal and cancer tissues

  • Evaluate correlation with clinical outcomes and cancer progression

  • CAMK2N2 plays a role in cell growth regulation when overexpressed in colon adenocarcinoma LoVo cells

• Signaling pathway investigation:

  • Examine CAMK2N2 expression in relation to calcium signaling components

  • Investigate CAMK2N2-CAMK2 interactions in cancer cells using co-immunoprecipitation

  • CAMK2 is known to regulate proliferation, differentiation, and survival of cancer cells

• Therapeutic response monitoring:

  • Evaluate changes in CAMK2N2 expression following treatment with anti-cancer agents

  • Investigate whether CAMK2N2 levels predict response to calcium signaling modulators

• Functional studies:

  • Use CAMK2N2 antibodies to monitor expression changes in knockdown/overexpression experiments

  • Correlate CAMK2N2 levels with cancer cell phenotypes (proliferation, migration, invasion)

How can I address non-specific binding issues when using CAMK2N2 antibodies?

Non-specific binding can significantly impact experimental outcomes. Consider these approaches:

• Antibody selection and optimization:

  • Choose antibodies validated specifically for your application and species

  • Test various antibody concentrations (recommended dilutions: IHC: 1:50-1:500; WB: 1:500-1:1000)

  • Consider monoclonal antibodies for higher specificity

• Blocking optimization:

  • Increase blocking time or concentration (typically 5% BSA or milk)

  • Use species-matched serum in blocking buffer

  • Add 0.1-0.3% Triton X-100 for membrane permeabilization in IHC/ICC

• Washing protocols:

  • Increase washing duration and frequency

  • Use PBS with 0.1% Tween-20 (PBST) for more stringent washing

• Antibody validation controls:

  • Include peptide competition assays

  • Use CAMK2N2 knockout/knockdown samples as negative controls

  • Pre-adsorb antibody with target tissue lysate from non-relevant species

What are the best methods to quantify CAMK2N2 protein levels in complex brain tissue samples?

Accurate quantification of CAMK2N2 in brain tissue requires specialized approaches:

• Western blot quantification:

  • Use appropriate loading controls (β-actin, GAPDH)

  • Include recombinant CAMK2N2 protein standards at known concentrations

  • Employ fluorescent secondary antibodies for wider linear range of detection

  • Normalize to total protein using stain-free technology

• ELISA-based quantification:

  • The GENLISA Human CAMK2N2 ELISA kit has a detection range of 0.156-10 ng/ml and sensitivity of 0.057 ng/ml

  • Validate standard curves with recombinant protein

  • Process samples consistently to minimize variation

• Mass spectrometry approaches:

  • Use targeted proteomics (SRM/MRM) for absolute quantification

  • Employ stable isotope-labeled peptide standards

  • Focus on unique peptides that distinguish CAMK2N2 isoforms

• Region-specific analysis:

  • Microdissect brain regions before analysis

  • Consider that CaM-KIINalpha shows stronger expression in frontal cortex and hippocampus, while CaM-KIINbeta shows stronger expression in cerebellum and hindbrain