CML24 Antibody

What is CML24 and why are antibodies against it important for research?

CML24 is a 161-amino acid, 16-kD calmodulin-like protein that shares approximately 40-41% identity with Arabidopsis calmodulins (CaMs). It contains four EF-hand motifs that enable calcium binding and subsequent conformational changes. CML24 (also known as TCH2) is part of the 50-member calmodulin-like (CML) Arabidopsis family that encodes proteins containing predicted calcium-binding EF-hand motifs .

Antibodies against CML24 are important research tools because they allow scientists to:

• Track CML24 protein expression and localization in different plant tissues

• Examine CML24's calcium-binding properties through mobility shift assays

• Investigate CML24's role in diverse stress responses such as touch, darkness, heat, cold, hydrogen peroxide, abscisic acid, and indole-3-acetic acid

• Study CML24's function in various physiological processes including flowering, germination, and salt tolerance

The ability to specifically detect CML24 amid other similar calcium-binding proteins makes this antibody particularly valuable for dissecting calcium signaling pathways in plants.

How is CML24 antibody typically produced for research applications?

CML24 antibody production follows a multi-step process requiring recombinant protein expression, purification, and immunization. The standard methodology includes:

• PCR amplification of the CML24 coding region using specific primers containing appropriate restriction sites

• Cloning into a bacterial expression vector (e.g., pET21a)

• Expression in bacterial cells (such as BL21(DE3)-RIL) induced with isopropylthio-β-galactoside

• Protein purification using calcium-dependent phenyl-Sepharose chromatography

• Verification of purified protein by SDS-PAGE

• Immunization of rabbits with the purified protein

• IgG purification from antiserum using commercially available kits

As reported in the literature, this approach produces antibodies with high specificity that can detect as little as 60 ng of purified CML24 in western blot applications, while showing no cross-reactivity with mammalian calmodulin, which shares 91% amino acid identity with Arabidopsis calmodulin .

What protein extraction methods are recommended when preparing samples for CML24 antibody detection?

For optimal CML24 detection, protein extraction methods should preserve protein integrity while maximizing yield. The recommended protocol is:

• Extract total plant protein using a lysis buffer containing 4% (w/v) SDS, 20% (v/v) glycerol, and 120 mM Tris, pH 6.8

• Determine protein concentration using a compatible assay such as the Pierce BCA kit

• Load 20-60 μg of protein per lane for SDS-PAGE separation

• Use 15% (w/v) SDS-polyacrylamide gels for optimal resolution of the low molecular weight CML24 protein

• Supplement Towbain's transfer buffer with 1-2 mM calcium chloride to assist with transfer of low molecular weight proteins

• Bake nitrocellulose membranes overnight at 65°C in a vacuum oven to enhance retention of CML24 protein

This extraction protocol has been validated for detecting endogenous CML24 in plant tissues and provides consistent results for quantitative analysis .

How can CML24 antibody be used to detect calcium-dependent conformational changes?

CML24 antibody can be leveraged to visualize calcium-dependent conformational changes through mobility shift assays. This advanced application requires specific experimental conditions:

• Prepare two sets of total plant protein samples

• Add either 5 mM EGTA (calcium chelator) or 5 mM calcium chloride to the samples

• Perform SDS-PAGE separation

• Transfer proteins to nitrocellulose membrane

• Probe with CML24 antibody

• Compare migration patterns between calcium-containing and EGTA-containing samples

The calcium-dependent mobility shift is observable as an increased electrophoretic mobility (faster migration) of CML24 in the presence of calcium compared to EGTA conditions. This phenomenon occurs because calcium binding induces a conformational change that persists even under the denaturing conditions of SDS-PAGE, suggesting profound structural alterations and high calcium affinity .

This technique provides valuable insights into CML24's function as a calcium sensor in plant cells and helps distinguish it from other calcium-binding proteins that may not exhibit similar conformational changes.

What controls and validation steps are essential when working with CML24 antibody?

When conducting experiments with CML24 antibody, researchers should implement several critical validation steps:

Research indicates the anti-CML24 antibody shows high specificity, failing to detect up to 20 μg of purified mammalian calmodulin despite the high sequence similarity between mammalian and Arabidopsis calmodulins. Additionally, the antibody does not cross-react with CML23, which shares 78% amino acid identity with CML24, further confirming its specificity .

How can researchers troubleshoot weak or absent CML24 antibody signals?

When experiencing detection issues with CML24 antibody, researchers should systematically address potential problems:

• Protein extraction efficiency:

  • Ensure complete tissue disruption using appropriate mechanical methods

  • Add protease inhibitors to prevent degradation

  • Verify protein concentration using multiple measurement methods

• Transfer optimization:

  • Supplement transfer buffer with 1-2 mM calcium chloride to improve transfer of low molecular weight proteins

  • Bake membranes overnight at 65°C in a vacuum oven to enhance protein retention

  • Consider using PVDF membranes instead of nitrocellulose for potentially better protein binding

• Antibody incubation conditions:

  • Optimize primary antibody concentration (2.4-3 μg/mL has been reported as effective)

  • Extend incubation time or adjust temperature

  • Try different blocking agents to reduce background while preserving specific signal

• Signal development:

  • Use enhanced chemiluminescence detection systems (like Pierce SuperSignal West Pico)

  • Increase exposure time for weak signals

  • Consider using more sensitive detection methods for low abundance proteins

• Expression levels:

  • Note that CML24 protein levels may not change significantly despite transcript level changes in response to stimuli, as reported in literature

  • Consider concentrating proteins through immunoprecipitation before western blotting

These troubleshooting steps address common technical challenges while acknowledging biological aspects of CML24 expression patterns.

What methodologies can be employed to study CML24's interaction with target proteins?

Investigating CML24's calcium-dependent interactions with target proteins requires specialized approaches:

• Co-immunoprecipitation (Co-IP):

  • Use purified CML24 antibody conjugated to protein A/G beads

  • Perform pull-downs in buffers containing either calcium or EGTA

  • Identify interacting partners through mass spectrometry

  • Validate interactions with reverse Co-IP using antibodies against putative partners

• Overlay assays:

  • Separate potential target proteins on SDS-PAGE

  • Transfer to membrane

  • Incubate with purified recombinant CML24 in calcium-containing buffer

  • Detect bound CML24 using CML24 antibody

  • Compare binding in calcium versus EGTA conditions

• Surface plasmon resonance:

  • Immobilize purified CML24 on sensor chip

  • Pass potential interacting proteins over the surface

  • Measure association and dissociation kinetics

  • Determine calcium dependency by comparing binding in calcium versus EGTA conditions

• Yeast two-hybrid screening with verification:

  • Use CML24 as bait to identify potential interactors

  • Verify interactions in planta using CML24 antibody-based Co-IP

  • Confirm calcium dependency of verified interactions

These methods leverage CML24 antibody's specificity to detect and analyze protein complexes that may form in response to calcium signaling events, providing insights into CML24's role as a calcium sensor in cellular processes.

How should researchers design experiments to investigate CML24's role in stress responses?

When investigating CML24's involvement in plant stress responses, researchers should consider:

• Stress treatment protocols:

  • Apply controlled stress conditions (touch, darkness, heat, cold, hydrogen peroxide, abscisic acid, indole-3-acetic acid)

  • Include appropriate time courses (minutes to hours) to capture early and late responses

  • Collect tissues from multiple organs to account for tissue-specific responses

• Expression analysis:

  • Measure both transcript levels via RT-PCR and protein levels via western blotting

  • Note that while transcripts may increase 2-15 fold in response to various stimuli, detectable changes in CML24 protein levels may not occur

  • Consider subcellular fractionation to detect potential redistribution rather than total expression changes

• Functional analysis:

  • Compare wild-type plants with CML24-underexpressing lines

  • Assess phenotypic differences in response to specific stressors

  • Document altered responses to abscisic acid, daylength perception, and salt tolerance

• Calcium dependency:

  • Perform calcium-binding mobility shift assays to determine if stress affects CML24's calcium-binding properties

  • Investigate potential post-translational modifications that might alter function without changing abundance

Research has shown that while CML24 transcript levels change significantly in response to diverse stimuli, protein abundance changes may not be detectable, suggesting potential regulation at the post-translational level or through rapid turnover mechanisms .

What are the critical parameters for optimizing western blotting with CML24 antibody?

Optimizing western blotting for CML24 detection requires attention to several critical parameters:

This optimized protocol accounts for the unique properties of CML24, including its low molecular weight, calcium-binding capacity, and potential for conformational changes that affect antibody recognition or protein retention on membranes .

How can CML24 antibody be used to investigate developmental regulation of calcium signaling?

CML24 antibody can be employed to study developmental regulation of calcium signaling through several methodological approaches:

• Developmental expression profiling:

  • Collect tissues at different developmental stages

  • Prepare protein extracts using the optimized extraction protocol

  • Perform western blotting with CML24 antibody

  • Quantify expression levels relative to appropriate loading controls

  • Correlate expression patterns with developmental events

• Tissue-specific localization:

  • Perform immunohistochemistry using CML24 antibody on tissue sections

  • Use fluorescently-labeled secondary antibodies for visualization

  • Compare localization patterns across different developmental stages

  • Correlate with sites of predicted mechanical stress, growing regions, vascular tissues, floral organs, stomata, trichomes, and hydathodes

• Co-expression with developmental markers:

  • Combine CML24 antibody detection with markers for specific developmental processes

  • Perform co-immunolocalization studies

  • Analyze temporal and spatial correlations between CML24 and developmental markers

• Functional studies in developmental mutants:

  • Compare CML24 expression and calcium-binding properties in wild-type versus developmental mutants

  • Analyze effects of altered CML24 expression on developmental timing and responses

  • Focus particularly on flowering induction, as CML24-underexpressing lines show defects in long-day induction of flowering

These approaches can reveal how CML24's calcium-sensing capability contributes to developmental processes, particularly in tissues with high calcium flux or mechanical stress.

What are the advantages and limitations of using CML24 antibody compared to other methods for studying calcium signaling?

Using CML24 antibody for calcium signaling research presents specific advantages and limitations researchers should consider:

Advantages:

• Enables direct detection of endogenous CML24 protein in complex biological samples

• Allows visualization of calcium-dependent conformational changes through mobility shift assays

• Provides specificity for CML24 over related calcium-binding proteins, including calmodulins and other CMLs

• Enables study of post-translational modifications that may affect function without altering transcript levels

• Can be used for multiple applications (western blotting, immunoprecipitation, immunohistochemistry)

Limitations:

• May not detect changes in CML24 protein levels even when transcript levels change significantly in response to stimuli

• Requires careful optimization of protein extraction and western blotting conditions

• Cannot directly visualize calcium binding in living cells in real-time

• Provides static snapshots rather than dynamic information about calcium signal transduction

• May not detect CML24 if it is complexed with interacting proteins that mask the epitope

Alternative methods such as fluorescent calcium indicators, genetically encoded calcium sensors, or transcript analysis provide complementary information but lack the specificity for studying CML24's unique role in calcium signal transduction.

How can researchers distinguish between CML24 and other closely related calmodulin-like proteins?

Distinguishing CML24 from related proteins requires careful experimental design and validation:

• Antibody specificity testing:

  • Confirm CML24 antibody does not cross-react with purified CaM or other related CMLs

  • Test against recombinant CML23, which shares 78% amino acid identity with CML24

  • Verify specific detection in wild-type versus CML24-underexpressing plants

• Protein characteristics differentiation:

  • Utilize distinct mobility patterns in calcium-shift assays

  • CML24 has a higher predicted isoelectric point (pI 4.78) than CaM (approximately 4.6)

  • CML24 contains more positively charged residues than CaM, with some replacing three glutamic acids found in CaM

  • CML24 has five glycines within the first six residues of the linker region, predicted to provide increased flexibility

• Genetic approaches:

  • Use CML24-specific gene silencing to confirm antibody specificity

  • Compare expression patterns in plants with altered expression of CML24 versus other CMLs

  • Verify through RT-PCR that silencing is specific to CML24 and does not affect related genes

Research has confirmed that anti-CML24 antibody does not recognize CML23 or CaM, supporting its use for specifically studying CML24 among the calmodulin and CML proteins .

What methodological approaches could advance our understanding of CML24's role in calcium signal transduction?

Future research on CML24 could benefit from several innovative methodological approaches:

• Proximity-dependent labeling techniques:

  • Express CML24 fused to promiscuous biotin ligases (BioID, TurboID)

  • Identify proteins in close proximity to CML24 under various stress conditions

  • Compare interactome in calcium-rich versus calcium-depleted environments

  • Validate key interactions using CML24 antibody-based co-immunoprecipitation

• Structural studies of CML24-target complexes:

  • Use CML24 antibody for immunoprecipitation of native complexes

  • Perform cryo-electron microscopy on purified complexes

  • Compare structures in calcium-bound versus calcium-free states

  • Correlate structural changes with functional outcomes

• In vivo calcium-dependent conformational sensors:

  • Develop FRET-based sensors incorporating CML24

  • Monitor real-time conformational changes in response to stimuli

  • Compare with biochemical results from CML24 antibody-based assays

  • Correlate with physiological responses

• Tissue-specific proteomics:

  • Use CML24 antibody for tissue-specific immunoprecipitation

  • Identify tissue-specific CML24 interactors or modifications

  • Compare interactome across developmental stages and stress conditions

  • Develop comprehensive model of CML24 function in different cellular contexts

These approaches would extend current knowledge by combining the specificity of CML24 antibody with advanced techniques for studying protein interactions, conformational dynamics, and tissue-specific functions.

How can researchers investigate potential post-translational modifications of CML24 using antibody-based approaches?

Investigating post-translational modifications (PTMs) of CML24 requires specialized antibody-based techniques:

• Two-dimensional gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Perform western blotting with CML24 antibody

  • Identify potential PTM-induced shifts in pI or molecular weight

  • Compare patterns across different stress conditions or developmental stages

• Phosphorylation-specific analysis:

  • Immunoprecipitate CML24 using CML24 antibody

  • Perform western blotting with phospho-specific antibodies

  • Alternatively, analyze immunoprecipitated protein by mass spectrometry

  • Compare phosphorylation status under different calcium concentrations or stress conditions

• PTM-enrichment strategies:

  • Perform immunoprecipitation with CML24 antibody

  • Enrich for specific modifications (phosphorylation, acetylation, etc.)

  • Identify modified residues by mass spectrometry

  • Create site-specific mutants to assess functional significance

• Calcium-dependent PTM analysis:

  • Compare PTM patterns in samples with calcium versus EGTA

  • Determine if calcium binding affects accessibility to modifying enzymes

  • Investigate if PTMs affect calcium-binding properties using mobility shift assays

These approaches could reveal how CML24's function is regulated beyond transcriptional control, potentially explaining the observation that transcript levels change significantly in response to stimuli while protein levels remain relatively constant .