COAC2 Antibody

What is COAC2 protein and what is its significance in plant research?

COAC2 (Graves disease carrier protein) is a protein-coding gene found in plant species including Arabidopsis thaliana. This protein has structural similarities to carrier proteins and may be involved in molecular transport mechanisms within plant cells. The protein is identified with the Uniprot number F4JU70 and has homologs in other plant species such as Solanum lycopersicum (tomato) where it is associated with gene ID LOC101266696 . Understanding COAC2's function is relevant for researchers studying plant molecular transport systems, cellular metabolism, and potentially stress responses. The antibody against this protein enables visualization and quantification of COAC2 in various experimental contexts, making it a valuable tool for plant biochemistry and molecular biology research.

Which research applications are validated for the COAC2 antibody?

The COAC2 antibody (CSB-PA561773XA01DOA) has been specifically validated for ELISA (Enzyme-Linked Immunosorbent Assay) applications . This validation ensures researchers can reliably use this antibody for quantitative detection of COAC2 protein in solution. While ELISA is the primary verified application, researchers commonly adapt antibodies for other immunological techniques following appropriate optimization protocols. It's important to note that this antibody is designated "For Research Use Only" and not for diagnostic or therapeutic procedures . Before attempting applications beyond ELISA, researchers should perform their own validation tests to confirm specificity and sensitivity in their specific experimental system.

What are the optimal storage conditions for maintaining COAC2 antibody integrity?

For maximum stability and retention of immunoreactivity, the COAC2 antibody should be stored at either -20°C or -80°C upon receipt . Repeated freeze-thaw cycles significantly degrade antibody quality and should be avoided. For optimal preservation:

Researchers should monitor antibody performance over time, as even properly stored antibodies may experience gradual loss of activity. Documentation of lot numbers and performance across experiments is recommended for longitudinal studies.

How can researchers optimize COAC2 antibody specificity validation for Arabidopsis studies?

Validating antibody specificity is crucial for generating reliable research data with COAC2 antibody. Since this antibody is raised against recombinant Arabidopsis thaliana COAC2 protein , researchers should implement multiple validation approaches:

• Western blot analysis comparing wild-type Arabidopsis with COAC2 knockout/knockdown lines to confirm absence of signal in mutant lines

• Peptide competition assays where pre-incubation of the antibody with excess purified COAC2 protein should abolish signal

• Immunoprecipitation followed by mass spectrometry to confirm the identity of pulled-down proteins

• Cross-reactivity testing with closely related proteins to ensure signal specificity

When presenting results, researchers should include all validation data alongside experimental findings to establish credibility. For negative results, thorough validation becomes even more critical to distinguish between true biological phenomena and technical limitations of the antibody.

What protocols are recommended for optimizing COAC2 antibody use in immunolocalization studies?

While the COAC2 antibody is primarily validated for ELISA , researchers can adapt it for immunolocalization studies with careful optimization:

• Fixation optimization: Test multiple fixatives (4% paraformaldehyde, glutaraldehyde combinations) as plant tissues often require specific fixation protocols to preserve antigenicity while maintaining cellular structure.

• Antigen retrieval: For formalin-fixed tissues, try citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) heat-mediated retrieval to expose epitopes.

• Blocking optimization: Test different blocking solutions (3-5% BSA, normal serum from the same species as secondary antibody) to reduce background.

• Antibody dilution series: Create a dilution series (typically 1:100 to 1:1000) to determine optimal signal-to-noise ratio.

• Signal amplification: For low-abundance proteins, consider tyramide signal amplification or quantum dot labeling.

The optimal protocol should produce specific labeling in wild-type samples with minimal background and absence of signal in negative controls (primary antibody omission, COAC2 knockout tissues).

How can COAC2 antibody be employed to study protein-protein interactions in plant stress responses?

Investigating COAC2 protein interactions during stress responses requires specialized immunological approaches:

• Co-immunoprecipitation (Co-IP): Use COAC2 antibody to pull down protein complexes from plant tissues under various stress conditions. This requires optimization of:

  • Lysis buffers (test multiple detergent concentrations)

  • Antibody concentration (typically 2-5 μg per mg of total protein)

  • Incubation conditions (4°C overnight with gentle rotation)

• Proximity ligation assay (PLA): This technique can visualize protein interactions in situ with spatial resolution:

  • Requires a second antibody against the suspected interaction partner

  • Provides single-molecule resolution of interaction events

  • Allows quantification of interaction frequency under different conditions

• Chromatin immunoprecipitation (ChIP) if COAC2 may have DNA-binding properties:

  • Optimize crosslinking conditions for plant tissues

  • Include appropriate controls (IgG, input samples)

What are common troubleshooting strategies for weak or absent COAC2 antibody signals in Western blots?

When experiencing poor signal in Western blots with COAC2 antibody, consider these methodological adjustments:

It's important to note that COAC2 antibody has been specifically validated for ELISA applications , so Western blot protocols will require additional optimization. If signals remain problematic after these adjustments, consider enriching for membrane fractions if COAC2 is predicted to be membrane-associated based on its possible carrier protein function.

How can researchers quantitatively assess COAC2 protein expression across different plant tissues?

For quantitative analysis of COAC2 expression across tissues, researchers should employ these methodological approaches:

• Quantitative ELISA:

  • Develop a standard curve using recombinant COAC2 protein

  • Process all tissue samples identically (extraction buffer, protein quantification)

  • Run technical triplicates and biological replicates (minimum n=3)

  • Include an internal control protein for normalization

• Western blot quantification:

  • Use housekeeping proteins (actin, tubulin) for normalization

  • Ensure signal is within linear detection range

  • Apply densitometry software (ImageJ) with consistent analysis parameters

  • Include a standard curve with recombinant protein if absolute quantification is needed

• High-content imaging:

  • When using immunofluorescence, standardize image acquisition parameters

  • Apply automated analysis algorithms to quantify signal intensity

  • Report results as mean fluorescence intensity normalized to cell count or tissue area

Reliable quantification requires statistical analysis of multiple biological replicates and consideration of tissue-specific extraction efficiency variations.

What protocols are recommended for adapting COAC2 antibody for chromatin immunoprecipitation (ChIP) studies?

If research suggests COAC2 may interact with DNA or chromatin-associated proteins, adapting the antibody for ChIP requires these specialized considerations:

• Crosslinking optimization:

  • Test formaldehyde concentrations (1-3%) and incubation times (10-20 minutes)

  • For plant tissues, vacuum infiltration improves crosslinking efficiency

  • Quench with glycine (final concentration 125 mM)

• Chromatin extraction and shearing:

  • Optimize sonication parameters for fragment sizes of 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

  • Pre-clear chromatin with protein A/G beads to reduce background

• Immunoprecipitation conditions:

  • COAC2 antibody amount: typically 2-5 μg per ChIP reaction

  • Include IgG control and input samples (5-10% of starting material)

  • Incubate overnight at 4°C with rotation

• Validation of ChIP efficiency:

  • Perform qPCR on known targets and negative control regions

  • Calculate percent input and fold enrichment over IgG control

Since the COAC2 antibody is raised in rabbit , protein A-based resins often provide optimal capture efficiency. The antibody's ability to recognize native, crosslinked COAC2 protein should be verified before proceeding with full ChIP-seq experiments.

How can COAC2 antibody be utilized to investigate protein degradation pathways in plant systems?

To investigate COAC2 protein turnover and degradation mechanisms, researchers can employ these specialized approaches:

• Cycloheximide chase assays:

  • Treat plants/cells with cycloheximide to inhibit protein synthesis

  • Harvest samples at multiple time points (0, 1, 3, 6, 12, 24 hours)

  • Perform Western blot using COAC2 antibody

  • Quantify protein levels relative to time zero and calculate half-life

• Proteasome inhibitor studies:

  • Treat samples with MG132 or other proteasome inhibitors

  • Compare COAC2 levels with and without inhibitor

  • Accumulation in inhibitor-treated samples suggests proteasomal degradation

• Ubiquitination detection:

  • Immunoprecipitate COAC2 using the antibody

  • Probe with anti-ubiquitin antibodies

  • Alternatively, co-express tagged ubiquitin and perform sequential immunoprecipitation

• Pulse-chase experiments:

  • Label newly synthesized proteins with 35S-methionine

  • Chase with non-radioactive medium

  • Immunoprecipitate COAC2 at various timepoints

  • Visualize by autoradiography and quantify degradation rate

When reporting results, include half-life calculations and statistical analysis of degradation rates under different conditions to robustly characterize COAC2 stability.

How might COAC2 antibody be utilized in comparative studies across plant species?

The COAC2 antibody is specifically raised against Arabidopsis thaliana COAC2 protein , but may have application in comparative studies with careful methodological considerations:

• Cross-reactivity assessment:

  • Perform sequence alignment analysis of COAC2 across species

  • Test antibody recognition in Western blots using protein extracts from multiple species

  • Quantify relative signal strength as indication of epitope conservation

• Epitope mapping:

  • If the epitope sequence is known, analyze conservation across species

  • For polyclonal antibodies, consider affinity purification against conserved epitopes

• Validation controls for cross-species studies:

  • Include positive controls (Arabidopsis samples)

  • Use genetic knockouts or knockdowns in test species when available

  • Employ peptide competition assays in each new species

Potential comparative studies could explore COAC2 homologs in Solanum lycopersicum and other plants, examining evolutionary conservation of expression patterns, subcellular localization, and protein-protein interactions. Such studies would contribute to understanding evolutionary conservation of COAC2 function across plant lineages.

What considerations are important when using COAC2 antibody in plant stress response studies?

When investigating COAC2's role in stress responses, methodological considerations include:

• Stress treatment standardization:

  • Define precise stress conditions (duration, intensity)

  • Include appropriate stress markers as positive controls

  • Document plant developmental stage and growth conditions

• Temporal analysis:

  • Perform time-course studies to capture dynamic changes

  • Include early (0-3h), intermediate (6-12h), and late (24-72h) timepoints

  • Correlate protein changes with transcriptional responses

• Subcellular fractionation:

  • Separate cellular compartments to track potential translocation

  • Verify fraction purity with compartment-specific markers

  • Quantify relative distribution across fractions

• Protein modification detection:

  • Assess post-translational modifications using phospho-specific stains

  • Use 2D gel electrophoresis to detect charge variants

  • Consider mass spectrometry to identify specific modifications

• Functional correlation:

  • Correlate COAC2 levels/modifications with physiological parameters

  • Compare wild-type and COAC2-modified plants under identical stress conditions

These approaches enable comprehensive characterization of COAC2's potential role in plant stress responses, particularly if it functions as a carrier protein involved in stress adaptation mechanisms.