CML13 Antibody

What is CML13 and what is its significance in plant molecular biology?

CML13 (Calmodulin-like protein 13) is a calcium sensor protein that belongs to the calmodulin-like protein family in plants. Unlike many other CMLs that exhibit tissue-specific or low basal expression, CML13 is notable for its high expression levels across various tissues and developmental stages in Arabidopsis thaliana . Recent research has established that CML13, along with its paralog CML14, functions as a novel light chain for myosin motor proteins, particularly class VIII and XI myosins . This discovery is significant because it reveals CML13's crucial role in cytoskeletal dynamics and intracellular transport, processes fundamental to plant growth, development, and responses to environmental stimuli.

CML13's significance stems from its involvement in:

• Calcium signaling cascades as a calcium sensor protein

• Cytoskeletal organization through interaction with myosin motor proteins

• Transcriptional regulation via interaction with CaM-binding transcriptional activators (CAMTAs)

• Protein-protein interactions with numerous proteins containing isoleucine-glutamine (IQ) domains

How does CML13 differ structurally and functionally from canonical calmodulin?

CML13 shares structural similarities with canonical calmodulin (CaM) but exhibits distinct functional properties that make it uniquely suited for specific biological roles. While both CML13 and CaM function as calcium sensor proteins and interact with IQ domain-containing proteins, CML13 demonstrates differential binding preferences and calcium-dependency patterns.

Key differences include:

• CML13 shows calcium-independent binding to IQ domains of myosins, whereas CaM binding to some targets is calcium-dependent

• CML13 exhibits preferential binding to specific IQ motifs (particularly IQ2 and IQ4) within myosin neck domains

• CML13 shows distinct residue preferences within IQ domains compared to CaM, as evidenced by mutation studies where "mutation of the conserved Iso in the IQ to an Ala produced little change to CML13 or CML14 interaction while abolishing CaM interaction to IQ1"

What are the most effective methods for generating specific antibodies against CML13?

Generating specific antibodies against CML13 requires careful consideration of antigen design and purification strategies to minimize cross-reactivity with other calmodulin-like proteins, particularly CML14, which shares high sequence similarity.

Recommended methodology:

• Recombinant protein expression: Express full-length CML13 or unique peptide sequences in bacterial systems (E. coli) using a pET or similar expression vector system with a 6xHis tag for purification.

• Epitope selection: Target unique regions that distinguish CML13 from CML14 and other CMLs, particularly focusing on the non-EF-hand domains or variable loops between EF-hands.

• Antibody production strategy: Employ a hybridoma technology approach similar to that used for CM313 production , involving:

  • Immunization of female BALB/c mice (aged 6-8 weeks) with purified recombinant CML13

  • Initial immunization with 50 μg protein mixed 1:1 with adjuvant

  • Follow-up immunizations with reduced dosage (25 μg) every two weeks

  • Selection of mice with favorable serum titers for final boost

  • Harvesting of splenocytes and fusion with myeloma cells

  • Screening of hybridoma clones for specific binding to CML13

• Validation: Confirm antibody specificity using multiple techniques:

  • Western blotting against recombinant CML13, CML14, and CaM

  • Immunoprecipitation followed by mass spectrometry

  • Immunohistochemistry in wild-type and cml13 knockout plants

How can I validate the specificity of my CML13 antibody and minimize cross-reactivity with CML14?

Given the high sequence similarity between CML13 and CML14, validating antibody specificity is crucial for experimental reliability.

Multi-step validation protocol:

• Side-by-side testing: Compare immunoreactivity against purified recombinant CML13, CML14, CaM, and other CMLs using ELISA and Western blotting.

• Genetic validation: Test antibody reactivity in wild-type, cml13 knockout, and cml13/cml14 double knockout plant tissues. Absence of signal in knockout lines confirms specificity.

• Competitive binding assays: Pre-incubate antibody with excess recombinant CML13 or CML14 before applying to samples. Specific antibodies should show signal reduction only when pre-incubated with CML13.

• Cross-adsorption: Purify antibody by adsorption against immobilized CML14 to remove cross-reactive antibodies, followed by affinity purification against CML13.

• Proteomic validation: Perform immunoprecipitation followed by mass spectrometry to identify all proteins pulled down by the antibody. A specific antibody should predominantly pull down CML13.

How can CML13 antibodies be utilized to study protein-protein interactions with myosins and other IQ domain-containing proteins?

CML13 antibodies serve as valuable tools for investigating the interactions between CML13 and its binding partners, particularly myosins and other IQ domain-containing proteins.

Recommended methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use CML13 antibodies conjugated to agarose or magnetic beads

  • Lyse plant tissues in a buffer containing appropriate protease inhibitors

  • Incubate lysate with antibody-conjugated beads

  • Wash extensively and elute bound proteins

  • Identify interacting partners by Western blotting or mass spectrometry

  • Compare results with control IgG to identify specific interactions

• Proximity ligation assay (PLA):

  • Fix plant tissues and permeabilize cells

  • Incubate with CML13 antibody and antibody against potential interacting protein

  • Apply oligonucleotide-linked secondary antibodies

  • Perform rolling circle amplification and fluorescent probe hybridization

  • Visualize interaction sites by fluorescence microscopy

  • This technique allows visualization of interactions in situ with high specificity

• Immunofluorescence co-localization:

  • Similar to the approach described in search result , where "CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein–myosin fusion proteins"

  • Use CML13 antibodies in combination with myosin-specific antibodies or fluorescent protein-tagged myosins

  • Analyze co-localization patterns under different conditions (calcium levels, developmental stages, stress responses)

What experimental designs are most effective for studying the role of CML13 in myosin light chain function?

Based on recent findings that CML13 functions as a myosin light chain , researchers can use CML13 antibodies to explore this functional relationship through several experimental approaches:

• In vitro actin motility assays:

  • Similar to those described in search result : "In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains"

  • Purify recombinant myosins (class VIII or XI) with and without bound CML13

  • Measure actin filament sliding velocity on myosin-coated surfaces

  • Compare activity of myosins with CML13 versus other light chains (CaM or CML14)

  • Assess the effects of calcium concentration on motility rates

• Structure-function analysis:

  • Use CML13 antibodies to pull down native myosin-CML13 complexes

  • Analyze the stoichiometry and binding sites using cross-linking and mass spectrometry

  • Map the specific interactions between CML13 and individual IQ domains

  • Correlate structural information with functional outcomes in motility assays

• Genetic complementation experiments:

  • Use CML13 antibodies to confirm protein expression in complementation lines

  • Follow the approach in search result : "cml13 T-DNA mutant exhibited a shortened primary root phenotype that was complemented by the wild-type CML13 and was similar to that observed in a triple myosin XI mutant (xi3KO)"

  • Quantify CML13 protein levels in different complementation lines

  • Correlate protein expression with phenotypic rescue

How can I use CML13 antibodies to distinguish between calcium-dependent and calcium-independent interactions?

Understanding the calcium dependency of CML13 interactions is crucial for elucidating its functional mechanisms. The search results indicate that "recombinant CaM, CML13, and CML14 exhibit calcium-independent binding to the IQ domains of myosin XIs" .

Methodological approach:

• Calcium-binding studies:

  • Perform immunoprecipitation with CML13 antibodies under varying calcium concentrations (0-10 mM)

  • Include calcium chelators (EGTA) in control experiments

  • Analyze bound proteins by mass spectrometry or Western blotting

  • Classify interacting partners as calcium-dependent or independent

• Structural analysis with calcium indicators:

  • Use fluorescent calcium indicators in combination with fluorescently-labeled CML13 antibodies

  • Monitor conformational changes in CML13 upon calcium binding

  • Correlate conformational changes with binding to different partners

• Calcium-gradient overlay assays:

  • Separate proteins by native gel electrophoresis

  • Transfer to membrane and overlay with recombinant CML13 in buffers containing different calcium concentrations

  • Detect bound CML13 using CML13 antibodies

  • Compare binding patterns across the calcium gradient

What approaches can resolve conflicting data between antibody-based detection and transcript-level expression of CML13?

Researchers often encounter discrepancies between protein detection and transcript abundance. For CML13, this is particularly relevant as search result indicates: "proteomic databases drew our attention to CML13 and CML14 as these paralogs were present at high levels and expressed broadly across tissues and developmental stages relative to most CMLs."

Resolution strategies:

• Integrated multi-omics approach:

  • Perform simultaneous protein quantification (using calibrated CML13 antibodies) and transcript analysis (qRT-PCR) on the same samples

  • Calculate protein-to-mRNA ratios across tissues and conditions

  • Plot correlation diagrams to identify conditions where discrepancies occur

  • Investigate post-transcriptional and post-translational mechanisms in these conditions

• Protein stability assessment:

  • Use cycloheximide chase assays with CML13 antibody detection to measure protein half-life

  • Compare degradation rates across tissues and conditions

  • Investigate ubiquitination and other post-translational modifications

• Translational efficiency analysis:

  • Perform polysome profiling to assess translational status of CML13 mRNA

  • Use CML13 antibodies to quantify newly synthesized protein (pulse labeling)

  • Correlate translational activity with protein abundance

What are the common technical challenges when using CML13 antibodies and how can they be overcome?

Researchers working with CML13 antibodies may encounter several challenges that can affect experimental outcomes.

Challenge 1: Cross-reactivity with CML14 and other CMLs

• Solution: Perform pre-adsorption against recombinant CML14 and other CMLs

• Include appropriate controls (cml13 knockout tissues) in all experiments

• Consider epitope-specific antibodies targeting unique regions of CML13

Challenge 2: Low signal intensity in certain tissues

• Solution: Optimize antigen retrieval methods for fixed tissues

• Employ signal amplification techniques (tyramide signal amplification)

• Use more sensitive detection methods (chemiluminescence for Western blots)

Challenge 3: Non-specific background in immunolocalization

• Solution: Increase blocking stringency (5% BSA, 0.3% Triton X-100)

• Include competing peptides in negative controls

• Optimize antibody concentration through titration experiments

How can I optimize immunoprecipitation protocols specifically for studying CML13-myosin interactions?

Optimizing immunoprecipitation (IP) for CML13-myosin complexes requires special consideration due to the nature of cytoskeletal proteins.

Optimized protocol:

• Sample preparation:

  • Harvest plant tissue and flash-freeze in liquid nitrogen

  • Grind tissue in liquid nitrogen to a fine powder

  • Extract proteins in a cytoskeleton-preserving buffer containing:

    • 50 mM PIPES (pH 6.9)

    • 5 mM MgCl₂

    • 5 mM EGTA

    • 0.5% Triton X-100

    • 10% glycerol

    • Protease inhibitor cocktail

  • Maintain samples at 4°C throughout processing

• Immunoprecipitation:

  • Pre-clear lysate with protein G beads

  • Incubate pre-cleared lysate with CML13 antibody (5 μg per 1 mg total protein)

  • Add protein G beads and incubate with gentle rotation for 3 hours at 4°C

  • Perform stringent washing (at least 5 washes) with buffer containing 150-300 mM NaCl

  • Elute bound proteins with glycine buffer (pH 2.5) or SDS sample buffer

• Analysis:

  • Separate eluted proteins by SDS-PAGE

  • Perform Western blotting with myosin-specific antibodies

  • Consider mass spectrometry analysis for unbiased identification of all interacting proteins

How should researchers interpret CML13 antibody signals in relation to developmental and stress-response studies?

Interpreting CML13 antibody signals requires consideration of the protein's varying roles across developmental stages and stress responses.

Interpretative framework:

• Developmental context:

  • According to search result , CML13 shows "high relative expression levels" and is "expressed broadly across tissues and developmental stages"

  • Establish baseline CML13 protein levels across key developmental stages

  • Normalize antibody signals to appropriate reference proteins for each tissue type

  • Consider the co-expression of interacting partners (myosins, IQDs, CAMTAs) when interpreting fluctuations

• Stress-response interpretation:

  • As a calcium sensor protein involved in signaling, CML13 may show altered expression or localization during stress

  • Compare CML13 levels before and after stress application

  • Correlate changes with calcium flux measurements

  • Distinguish between changes in total protein (Western blot) versus localization (immunofluorescence)

• Quantitative analysis:

  • Use calibration curves with recombinant CML13 for absolute quantification

  • Apply digital image analysis software for consistent quantification

  • Present data as fold-change relative to appropriate controls

What statistical approaches are most appropriate for analyzing CML13 quantification data from antibody-based experiments?

Recommended statistical approaches:

How can CML13 antibody-based approaches be complemented with genetic and biochemical techniques?

Integrating multiple technical approaches provides a more comprehensive understanding of CML13 function.

Integrated research strategy:

• Genetic approaches:

  • Use CML13 antibodies to validate protein absence in knockout or knockdown lines

  • Compare phenotypes between cml13 single mutants and cml13/cml14 double mutants to assess functional redundancy

  • Follow the approach in search result : "Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant"

• Biochemical approaches:

  • Combine antibody detection with activity assays for calcium binding

  • Use split-luciferase complementation assays as described in search result : "in planta split luciferase (SL) protein-protein interaction assay"

  • Correlate protein-protein interactions detected by antibodies with functional outcomes in activity assays

• Structural biology integration:

  • Use antibody-based purification of native complexes for structural studies

  • Compare results with recombinant protein studies

  • Correlate structural features with functional outcomes

What novel applications of CML13 antibodies might advance our understanding of plant calcium signaling networks?

Several innovative applications of CML13 antibodies could provide new insights into plant calcium signaling.

Novel applications:

• Proximity-dependent biotin identification (BioID):

  • Create fusion proteins of CML13 with a biotin ligase

  • Use CML13 antibodies to confirm expression and localization

  • Identify proteins in proximity to CML13 during different calcium signaling events

  • This approach can reveal transient or weak interactions not captured by traditional co-IP

• Single-molecule tracking:

  • Label CML13 antibodies with quantum dots or other bright, photostable fluorophores

  • Track individual CML13 molecules in living cells using super-resolution microscopy

  • Analyze diffusion coefficients and residence times at cellular structures

  • Correlate mobility with calcium concentration and cellular responses

• Tissue-specific interactome analysis:

  • Perform tissue-specific immunoprecipitation using CML13 antibodies

  • Compare interacting partners across different tissues and developmental stages

  • Create tissue-specific interactome maps to understand context-dependent functions of CML13

  • Integrate with transcriptomic and phosphoproteomic data for comprehensive signaling network models