CAMTA3 Antibody

What is CAMTA3 and why do researchers need antibodies against it?

CAMTA3 is a plant transcription factor that functions as a repressor of immunity-related genes but an activator of cold-induced genes in plants . It contains multiple functional domains including a CG-1 DNA binding domain that recognizes CGCG elements in target gene promoters .

Researchers need CAMTA3 antibodies to:

• Detect protein expression levels in different tissues and under various stress conditions

• Examine post-translational modifications, particularly phosphorylation induced by bacterial elicitors like flg22

• Perform chromatin immunoprecipitation to identify DNA binding sites

• Study subcellular localization changes in response to stimuli

• Investigate protein-protein interactions

CAMTA3 is particularly important because mutants of camta3 exhibit autoimmune phenotypes including stunted growth, chlorosis, leaf lesions, constitutive expression of defense genes, and enhanced resistance to pathogens .

How can researchers validate the specificity of CAMTA3 antibodies?

Validating antibody specificity is critical for reliable CAMTA3 research. Recommended validation methods include:

• Western blot analysis using:

  • Wild-type plant tissue compared with camta3 mutant tissue (negative control)

  • Recombinant CAMTA3 protein as positive control

  • CAMTA3 overexpression lines

• Immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before using it in applications

• Cross-reactivity testing against other CAMTA family members (CAMTA1, CAMTA2)

In published studies, researchers have used immunoblot analysis to confirm the size and purity of CAMTA3 protein variants, detecting a single band of expected size (~24 kDa) for the CG-1 domain of CAMTA3 .

What protein extraction methods optimize CAMTA3 detection?

Effective protein extraction is crucial for reliable CAMTA3 detection:

• Buffer components for complete extraction:

  • High salt concentration (300-500 mM NaCl) to extract nuclear transcription factors

  • Non-ionic detergents (0.5-1% NP-40 or Triton X-100) to disrupt membranes

  • Glycerol (10-20%) to stabilize proteins

  • DTT (1-5 mM) to maintain reducing conditions

• Essential protease and phosphatase inhibitors:

  • Complete protease inhibitor cocktail to prevent degradation

  • Phosphatase inhibitors (sodium fluoride, sodium orthovanadate) to preserve phosphorylation states

  • For studying degradation: include proteasome inhibitors (MG132)

• Extraction procedure:

  • Maintain samples at 4°C throughout extraction

  • Use gentle homogenization followed by centrifugation

  • For nuclear CAMTA3: first isolate nuclei, then extract with high-salt buffer

When studying phosphorylated forms of CAMTA3, as seen after flg22 treatment, phosphatase inhibitors are particularly critical to preserve the phospho-mobility shift that can be detected on Western blots .

What expression patterns of CAMTA3 have been observed in plant tissues?

CAMTA3 expression shows distinctive patterns that researchers should consider when designing experiments:

• Basal expression:

  • Present in various tissues with primarily nuclear localization as expected for a transcription factor

  • Functions as a repressor of defense-related genes under normal conditions

• Stress-induced expression changes:

  • Transcript levels increase rapidly (within 30-60 minutes) after flg22 treatment and return to near basal levels after ~4 hours

  • Cold treatment induces expression of CAMTA3-regulated genes without triggering its degradation

• Subcellular dynamics:

  • Under normal conditions: predominantly nuclear localization

  • After pathogen elicitor (flg22) treatment: undergoes nuclear export to the cytoplasm followed by degradation

This dual expression pattern reflects CAMTA3's role as both a repressor of immunity genes and an activator of cold-responsive genes, allowing it to function in cross-talk between these stress response pathways.

How can researchers detect CAMTA3-DNA interactions using antibody-based methods?

CAMTA3 binds to specific DNA sequences containing CGCG elements through its CG-1 domain . Several antibody-based approaches can detect these interactions:

• Chromatin Immunoprecipitation (ChIP):

  • Cross-link proteins to DNA in planta using 1% formaldehyde (10 minutes)

  • Isolate and sonicate chromatin to 200-500 bp fragments

  • Immunoprecipitate with CAMTA3 antibody

  • Analyze bound DNA by qPCR targeting known CAMTA3-regulated genes (e.g., EDS1, NDR1, EIN3)

• Electrophoretic Mobility Shift Assay (EMSA) with supershift:

  • Generate labeled DNA probes containing CAMTA3 binding sites from promoters like PDF1.4

  • Incubate with nuclear extracts

  • Add CAMTA3 antibody to create a supershift, confirming CAMTA3's presence in the DNA-protein complex

  • Resolve on native polyacrylamide gel (5%)

• DNA affinity purification with immunodetection:

  • Immobilize DNA containing CAMTA3 binding sites

  • Incubate with nuclear extracts

  • Wash and elute bound proteins

  • Detect CAMTA3 by Western blot

The binding specificity of CAMTA3 has been demonstrated using EMSA, where purified wild-type CG-1 domain showed binding to a PDF1.4 promoter probe in a concentration-dependent manner, with binding detectable with as little as 25 ng protein .

What methods can detect CAMTA3 phosphorylation after flg22 treatment?

CAMTA3 undergoes phosphorylation upon treatment with bacterial flg22 elicitor, showing a distinctive phospho-mobility shift . Methods to study this modification include:

• Phospho-mobility shift detection:

  • Treat plants with flg22 (time course from 15-120 minutes)

  • Extract proteins with phosphatase inhibitors

  • Perform SDS-PAGE using lower percentage gels (6-8%)

  • Western blot with CAMTA3 antibody to detect the mobility shift

• Kinase pathway analysis:

  • Use kinase mutants (mpk3/mpk6) to determine pathway dependence

  • Test phospho-null CAMTA3 mutants that cannot be phosphorylated by MAPKs

  • Compare with constitutively-active calcium-dependent protein kinase (CPK5) treatment

• Phosphatase treatment controls:

  • Treat duplicate protein samples with lambda phosphatase

  • Compare migration patterns to confirm phosphorylation

Research has shown that flg22 treatment induces CAMTA3 phosphorylation through at least two independent pathways: the MAPK pathway (MPK3/MPK6) and potentially a CPK5-mediated pathway . Interestingly, a phospho-null CAMTA3 that cannot be phosphorylated by MAPKs still shows nuclear export after flg22 treatment, suggesting multiple regulatory mechanisms .

How can researchers study CAMTA3 translocation between cellular compartments?

CAMTA3 undergoes nuclear export following flg22 treatment, a critical step in its regulation . To study this translocation:

• Subcellular fractionation approach:

  • Treat plants with flg22 (time course from 0-120 minutes)

  • Isolate nuclear and cytoplasmic fractions

  • Perform Western blotting with CAMTA3 antibody

  • Include markers for nuclear (histone H3) and cytoplasmic (GAPDH) fractions

• Live cell imaging with fluorescent fusion proteins:

  • Generate plants expressing CAMTA3-GFP under native promoter

  • Perform time-lapse imaging after flg22 treatment

  • Quantify nuclear/cytoplasmic fluorescence ratio over time

• Mechanism investigation:

  • Test effects of protein kinase inhibitors on translocation

  • Investigate nuclear export machinery using inhibitors (leptomycin B)

  • Study CAMTA3 mutants to identify nuclear localization/export signals

• Data quantification:

  • Calculate the nuclear/cytoplasmic ratio of CAMTA3 at different timepoints

  • Correlate translocation with downstream gene expression changes

Research has shown that phosphorylation by MPK3/MPK6 triggers CAMTA3 nuclear export, followed by destabilization, presumably to remove CAMTA3 from target promoters and de-repress expression of defense genes .

What approaches can quantify changes in CAMTA3 protein levels during stress responses?

Accurately quantifying CAMTA3 protein dynamics during stress responses requires:

• Western blot quantification:

  • Collect samples at multiple timepoints after treatment (0-240 minutes)

  • Perform Western blot with CAMTA3 antibody

  • Use internal loading controls (actin/tubulin or total protein stain)

  • Analyze band intensity using densitometry software

• Experimental design considerations:

  • Include proteasome inhibitors in parallel samples to block degradation

  • Compare transcript and protein levels simultaneously

  • Monitor both nuclear and cytoplasmic fractions

• Data analysis approaches:

  • Calculate relative protein abundance normalized to controls

  • Generate time-course curves showing protein dynamics

  • Correlate protein changes with transcriptional effects on target genes

How does the DNA binding domain of CAMTA3 function?

The CG-1 domain is responsible for CAMTA3's DNA binding activity. Recent studies have characterized its function:

• Recognition sequence:

  • Binds specifically to CGCG elements in target gene promoters

  • Well-characterized binding sites include those in defense genes such as PDF1.4, containing the core "CGCG" element

• Structural features:

  • Contains several highly conserved amino acids across plant and animal CAMTAs

  • Critical amino acid residues have been identified as necessary for DNA binding activity

• Functional significance:

  • DNA binding activity is essential for CAMTA3's role in mediating plant immune responses

  • Mutants with altered CG-1 domain lose the ability to bind DNA and fail to complement camta3 mutant phenotypes

EMSA studies have shown that wild-type CG-1 domain binds to the PDF1.4 promoter in a concentration-dependent manner, with binding detectable with as little as 25 ng protein . This binding is specific and can be competed with unlabeled DNA containing the same binding site.

What approaches can analyze mutant forms of CAMTA3?

Mutational analysis has been essential for understanding CAMTA3 function:

• Generation of CAMTA3 mutants:

  • Site-directed mutagenesis targeting conserved amino acids in the CG-1 domain

  • Q5 site-directed mutagenesis kit with primers designed using the NEB Base changer web portal

  • Expression in E. coli for protein purification or in plants for functional studies

• In vitro analysis of mutant proteins:

  • Purification of His-tagged wild-type and mutant proteins

  • EMSA to assess DNA binding capabilities

  • Western blot using anti-His antibodies to confirm expression and size

• In vivo functional evaluation:

  • Generation of transgenic plants expressing CG-1 mutants in camta3 background

  • Assessment of phenotypic rescue

  • Analysis of target gene expression through qRT-PCR

Recent studies demonstrated that mutations in conserved amino acids in the CG-1 domain abolished DNA binding activity in vitro and failed to rescue the camta3 mutant phenotype in transgenic plants , proving that DNA binding is essential for CAMTA3 function.

How do researchers analyze the transcriptional activity of CAMTA3?

CAMTA3 exhibits complex transcriptional activity that can be analyzed through:

• Reporter gene assays:

  • Transient expression systems using reporter constructs with CAMTA3 binding sites

  • RSRE (Rapid Stress Response Element)-driven reporter gene expression

  • Co-expression of wild-type or mutant CAMTA3 as effector constructs

• Target gene expression analysis:

  • qRT-PCR of known CAMTA3-regulated genes:

    • Repressed genes: defense-related genes (PR1, EDS1)

    • Activated genes: cold-responsive genes (CBF2)

  • Comparison between wild-type, camta3 mutants, and complementation lines

• Chromatin immunoprecipitation:

  • ChIP-qPCR targeting specific promoters containing CGCG elements

  • Analysis of CAMTA3 occupancy at target genes under different conditions

Transient assays have shown that conserved residues in the CG-1 domain are essential for CAMTA3 function in activating RSRE-driven reporter gene expression . Additionally, transgenic lines expressing CG-1 mutants in the camta3 background failed to restore the expression of CAMTA3 downstream target genes , confirming the importance of DNA binding for transcriptional regulation.

How can researchers resolve weak signal problems with CAMTA3 antibodies?

When facing weak signals in CAMTA3 detection, researchers should consider:

• Protein extraction optimization:

  • Use stronger lysis conditions for complete extraction from nuclear fraction

  • Increase protease inhibitor concentration to prevent degradation

  • Include phosphatase inhibitors to preserve modified forms

• Antibody optimization:

  • Test different antibody concentrations (consider 1:500 to 1:2000 range)

  • Try longer incubation times (overnight at 4°C)

  • Use signal enhancement systems (HRP-conjugated secondary antibodies with enhanced chemiluminescence)

• Technical adjustments:

  • Increase protein loading (50-100 μg total protein)

  • Reduce washing stringency or duration

  • Use PVDF membranes instead of nitrocellulose for stronger protein binding

• Consider protein dynamics:

  • CAMTA3 undergoes degradation after flg22 treatment

  • Include proteasome inhibitors if studying phosphorylated forms

  • Carefully time sample collection to capture protein before degradation

For recombinant proteins, published protocols have successfully used anti-His tag monoclonal antibody at 1:2000 dilution for immunoblot detection of CAMTA3 variants .

What factors affect detection of phosphorylated vs. non-phosphorylated CAMTA3?

Distinguishing phosphorylated CAMTA3 requires specific considerations:

• Sample preparation adjustments:

  • Include comprehensive phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Prepare parallel samples treated with lambda phosphatase as controls

  • Use Phos-tag acrylamide gels for enhanced separation of phosphorylated forms

• Gel electrophoresis optimization:

  • Use lower percentage acrylamide gels (6-8%) for better separation

  • Run gels at lower voltage (80-100V) for improved resolution

  • Extend running time to enhance separation of closely migrating bands

• Experimental controls:

  • Include samples from kinase mutants (mpk3/mpk6)

  • Test phospho-null CAMTA3 mutants that cannot be phosphorylated by MAPKs

  • Compare with constitutively-active CPK5 treatment samples

Research has shown that flg22 treatment induces a phospho-mobility shift in CAMTA3 that can be detected via Western blot . Importantly, at least two independent signaling pathways contribute to CAMTA3 phosphorylation - the MAPK pathway and potentially a CPK5-dependent pathway .

How can cross-talk between immune and cold response pathways be studied using CAMTA3 antibodies?

CAMTA3 functions at the intersection of immune and cold response pathways, offering unique research opportunities:

• Comparative analysis approach:

  • Compare protein modifications after pathogen elicitors vs. cold treatment

  • Monitor subcellular localization under different stresses

  • Track binding to different target promoters (defense vs. cold-responsive genes)

• Dual stress experiments:

  • Apply sequential stresses (cold followed by pathogen elicitors or vice versa)

  • Monitor CAMTA3 protein levels, modifications, and localization

  • Correlate with expression of both immune and cold-responsive genes

• Mutation-based approaches:

  • Study phospho-site mutants in both stress conditions

  • Use domain deletion mutants to identify regions required for specific responses

  • Compare binding to different target promoters

Research shows that CAMTA3 undergoes phosphorylation and degradation specifically in response to immune triggers (flg22) but not cold stress . This selective regulation may explain how CAMTA3 can act as both a repressor of immunity genes and an activator of cold-responsive genes, allowing plants to appropriately balance responses to different environmental challenges.