At2g34840 Antibody

What is the At2g34840 gene and its function in Arabidopsis thaliana?

At2g34840 encodes the Coatomer epsilon subunit protein in Arabidopsis thaliana (thale cress). It is a protein-coding gene with synonyms F19I3.7 and F19I3_7 in the Arabidopsis genome . The gene has been assigned the Entrez Gene ID 818049 and corresponds to UniProt accession number O64748 .

As a component of the COPI coatomer complex, the epsilon subunit plays a crucial role in vesicle-mediated protein transport within the cell, particularly in retrograde transport from the Golgi apparatus to the endoplasmic reticulum. This protein is part of the cellular machinery responsible for maintaining proper protein trafficking and organelle integrity in plant cells. The gene is conserved across various species, indicating its fundamental importance in eukaryotic cell biology.

What specific applications is At2g34840 antibody validated for?

The At2g34840 antibody has been validated for several experimental applications in plant research. According to the product specifications, it has been tested and confirmed effective for ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) applications . These techniques allow researchers to detect and quantify the Coatomer epsilon subunit protein in various experimental contexts.

For Western blot applications, the antibody enables detection of the target protein in complex mixtures of cellular proteins after separation by gel electrophoresis. This application is particularly useful for studying protein expression levels, molecular weight confirmation, and post-translational modifications. The ELISA application provides a sensitive method for quantifying the protein in solution, which is valuable for comparative studies of protein expression under different experimental conditions.

What are the optimal storage conditions and handling precautions for At2g34840 antibody?

For maximum stability and activity retention, At2g34840 antibody should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be avoided as they can significantly degrade antibody performance and specificity. If frequent use is anticipated, aliquoting the antibody into smaller volumes before freezing is recommended to minimize freeze-thaw cycles.

The antibody is supplied in liquid form with a specific storage buffer composition: 50% Glycerol, 0.01M PBS at pH 7.4, and 0.03% Proclin 300 as a preservative . This formulation helps maintain antibody stability during storage. When handling the antibody, researchers should use sterile technique and avoid contamination. For short-term use during experiments, keeping the antibody on ice is advised to prevent protein degradation.

How should researchers validate the specificity of At2g34840 antibody in their experimental systems?

Validating antibody specificity is crucial for reliable experimental outcomes. For At2g34840 antibody, researchers should implement multiple validation strategies:

• Positive and negative controls: Include wild-type Arabidopsis samples (positive control) and either knockout/knockdown lines of At2g34840 or non-plant samples (negative controls).

• Blocking peptide competition assay: Pre-incubate the antibody with the immunizing peptide/recombinant protein before application to demonstrate binding specificity.

• Multiple detection methods: Confirm results using different techniques (e.g., both Western blot and immunoprecipitation) to strengthen confidence in specificity.

• Molecular weight verification: The detected band in Western blot should correspond to the predicted molecular weight of the Coatomer epsilon subunit.

Researchers should note that this antibody was generated using recombinant Arabidopsis thaliana At2g34840 protein as the immunogen , which provides specificity for the target but may also introduce the possibility of recognizing closely related epitopes in homologous proteins.

What experimental controls are essential when using At2g34840 antibody?

When designing experiments with At2g34840 antibody, several controls are essential for result validation:

• Loading control: For Western blots, include detection of a housekeeping protein (e.g., actin or tubulin) to normalize protein loading across samples.

• Negative control: Include samples from At2g34840 knockout/knockdown lines or use secondary antibody alone to identify non-specific binding.

• Positive control: Use samples with known expression of At2g34840, such as wild-type Arabidopsis tissues where Coatomer epsilon subunit is well-characterized.

• Recombinant protein standard: Include purified recombinant At2g34840 protein at known concentrations to create a standard curve for quantification purposes.

• Tissue-specific controls: When examining expression patterns, include multiple tissue types to confirm expected distribution patterns of the protein.

These controls help ensure that experimental observations are specific to the Coatomer epsilon subunit and not artifacts of the detection system or sample preparation.

What are the critical optimization steps for using At2g34840 antibody in Western blot applications?

Optimizing Western blot protocols for At2g34840 antibody requires attention to several key parameters:

• Sample preparation: Plant tissues contain compounds that can interfere with antibody binding. Use extraction buffers containing appropriate protease inhibitors and reducing agents to maintain protein integrity. Consider adding 2% polyvinylpolypyrrolidone (PVPP) to remove phenolic compounds.

• Blocking conditions: Test different blocking agents (BSA, non-fat milk, commercial blockers) at various concentrations (3-5%) to minimize background while preserving specific signal. The antibody's storage buffer contains 50% glycerol and PBS at pH 7.4 , so consider compatibility with your blocking solution.

• Antibody dilution optimization: Conduct a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) to determine the optimal concentration that provides specific signal with minimal background. Given this is an affinity-purified polyclonal antibody , lower dilutions may be needed compared to monoclonal alternatives.

• Incubation conditions: Test both room temperature incubation (1-2 hours) and 4°C overnight incubation to determine optimal signal-to-noise ratio. The temperature sensitivity of epitope recognition can affect binding efficiency.

• Detection system selection: For low abundance proteins like coatomer components, enhanced chemiluminescence (ECL) or fluorescent secondary antibodies may provide better sensitivity than colorimetric detection methods.

Researchers should document optimization steps methodically, changing only one parameter at a time to identify the ideal conditions for their specific experimental system.

How can researchers quantitatively assess Coatomer epsilon subunit expression using At2g34840 antibody?

Quantitative assessment of the Coatomer epsilon subunit requires rigorous methodological approaches:

• Quantitative Western blotting: Use a dilution series of recombinant At2g34840 protein to create a standard curve. Analyze band intensities with densitometry software, normalizing to loading controls. Ensure exposures remain in the linear range of detection.

• ELISA quantification: Develop a sandwich ELISA using At2g34840 antibody as the capture or detection antibody. Similar to IgG detection methods described in the literature , prepare standard curves using purified recombinant protein.

• Statistical validation: Perform multiple biological replicates (minimum n=3) and technical replicates to enable statistical analysis. Calculate coefficients of variation to ensure assay reliability, aiming for intra-assay CV <5% and inter-assay CV <10%, comparable to standards in immunoglobulin quantification (3.4% and 2.9% for intra- and inter-assay, respectively) .

• Data normalization: For comparative studies, normalize expression data to appropriate reference proteins or total protein content using methods such as Ponceau S staining or housekeeping protein detection.

Sample ELISA procedure for At2g34840 quantification could follow protocols similar to those used for immunoglobulin detection, adapting the capture and detection antibodies specifically for the Coatomer epsilon subunit.

What methodological approaches can resolve common issues in At2g34840 antibody specificity?

Resolving specificity issues with At2g34840 antibody requires systematic troubleshooting:

• Cross-reactivity assessment: Test the antibody against recombinant proteins from related coatomer subunits to identify potential cross-reactivity. Particularly important is testing against AT1G30630, another Arabidopsis Coatomer epsilon subunit homolog identified in comparative sequence analyses .

• Epitope mapping: If persistent cross-reactivity occurs, consider epitope mapping to identify the specific regions recognized by the polyclonal antibody. This information can help predict potential cross-reactive proteins.

• Pre-adsorption protocols: For polyclonal antibodies with minor cross-reactivity, pre-adsorb the antibody with proteins or extracts containing the cross-reactive epitopes but lacking the target protein.

• Immunodepletion: If specific bands persist in knockout controls, perform immunodepletion experiments to confirm whether these represent genuine cross-reactivity or non-specific binding.

• Alternative detection methods: Validate findings using orthogonal approaches such as mass spectrometry-based protein identification of immunoprecipitated material.

Implementing these methodological approaches can significantly enhance the specificity of experiments using At2g34840 antibody and increase confidence in experimental outcomes.

How can At2g34840 antibody be effectively used in co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) with At2g34840 antibody requires careful methodological considerations:

• Antibody immobilization: The At2g34840 antibody is an IgG isotype , making it suitable for immobilization on Protein A/G resins. Covalent cross-linking to the resin using dimethyl pimelimidate can prevent antibody leaching and contamination of samples with heavy and light chains.

• Extraction buffer optimization: For membrane-associated proteins like coatomer components, test different detergents (e.g., CHAPS, digitonin, NP-40) at various concentrations to solubilize protein complexes while maintaining native interactions. Buffer compositions similar to those used in antigen-antibody interaction studies for other complex systems can serve as starting points .

• Control experiments: Include IgG isotype controls from the same species (rabbit) to identify non-specific binding. Additionally, perform reverse Co-IP experiments when possible, using antibodies against suspected interaction partners.

• Washing stringency optimization: Determine the optimal balance between stringent washing (to reduce non-specific binding) and gentle conditions (to preserve genuine interactions). Test buffers with varying salt concentrations (150-500 mM NaCl) and detergent levels.

• Elution strategies: Compare different elution methods, including low pH, high salt, competitive elution with immunizing peptide, or direct boiling in SDS sample buffer to determine which provides the cleanest and most complete recovery of protein complexes.

By methodically optimizing these parameters, researchers can effectively use At2g34840 antibody to study the interaction network of the Coatomer epsilon subunit in vesicle trafficking pathways.

What considerations are important when using At2g34840 antibody for subcellular localization studies?

When employing At2g34840 antibody for immunolocalization of the Coatomer epsilon subunit:

• Fixation protocol optimization: Test different fixatives (paraformaldehyde, glutaraldehyde, methanol) and concentrations to preserve both protein antigenicity and cellular architecture. The epitope recognized by this polyclonal antibody may be sensitive to specific fixation conditions.

• Permeabilization methods: Optimize membrane permeabilization using different detergents (Triton X-100, saponin, digitonin) at various concentrations and incubation times to ensure antibody access to intracellular compartments without destroying membrane structures important for coatomer localization.

• Antigen retrieval techniques: If fixation reduces antibody binding, evaluate antigen retrieval methods such as heat-induced epitope retrieval or enzymatic treatment to expose epitopes while maintaining cellular structure.

• Signal amplification strategies: For low-abundance proteins, consider signal amplification methods such as tyramide signal amplification or quantum dot-conjugated secondary antibodies to enhance detection sensitivity.

• Co-localization controls: Include markers for relevant compartments (Golgi apparatus, ER, COPI vesicles) to confirm the expected subcellular distribution pattern. Calculation of co-localization coefficients (Pearson's or Mander's) provides quantitative assessment of spatial relationships.

• Super-resolution techniques: Consider advanced imaging methods like structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) to resolve coatomer-coated vesicles beyond the diffraction limit of conventional microscopy.

These methodological considerations help ensure reliable and informative subcellular localization data when using At2g34840 antibody for immunofluorescence or immunoelectron microscopy studies.

What is the recommended protocol for Western blot using At2g34840 antibody?

The following detailed protocol is recommended for Western blot application of At2g34840 antibody:

• Sample preparation:

  • Grind Arabidopsis tissue in liquid nitrogen

  • Extract proteins in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, protease inhibitor cocktail

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Determine protein concentration using Bradford assay

• SDS-PAGE separation:

  • Load 20-50 μg total protein per lane

  • Separate proteins on 10-12% polyacrylamide gel

  • Include molecular weight markers and positive control samples

• Transfer procedure:

  • Transfer proteins to PVDF membrane (0.45 μm pore size)

  • Use semi-dry or wet transfer at 100V for 1 hour or 30V overnight at 4°C

• Immunoblotting:

  • Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with At2g34840 antibody at 1:1000 dilution in blocking buffer overnight at 4°C

  • Wash 3 × 10 minutes with TBST

  • Incubate with HRP-conjugated goat anti-rabbit IgG (1:5000) for 1 hour at room temperature

  • Wash 5 × 5 minutes with TBST

• Detection:

  • Apply enhanced chemiluminescence substrate

  • Expose to X-ray film or capture images using digital imaging system

  • Perform densitometric analysis for quantification

This protocol incorporates elements from standard immunoblotting procedures adapted specifically for plant tissue samples and the properties of the At2g34840 antibody.

How can researchers optimize ELISA protocols for At2g34840 detection?

ELISA optimization for At2g34840 detection should follow these methodological steps:

• Plate coating optimization:

  • Test different coating buffers (carbonate buffer pH 9.6, PBS pH 7.4)

  • Optimize coating concentration (1-10 μg/ml) of capture antibody

  • Determine optimal coating time (2 hours at 37°C or overnight at 4°C)

• Blocking parameter optimization:

  • Compare blocking agents (BSA, non-fat milk, commercial blockers)

  • Test different concentrations (1-5%) and incubation times

• Sample preparation:

  • Develop extraction protocols that minimize interference from plant compounds

  • Determine appropriate sample dilutions (10-fold serial dilutions)

  • Include purified recombinant At2g34840 protein standards (0.2-12.5 ng/100 μL range)

• Detection system optimization:

  • For direct ELISA: use directly labeled At2g34840 antibody

  • For sandwich ELISA: use a second detection antibody against a different epitope

  • Test different enzyme conjugates (HRP or alkaline phosphatase)

  • Optimize substrate development time (15-30 minutes)

• Validation:

  • Calculate coefficients of variation for intra- and inter-assay reproducibility

  • Aim for values <10%, comparable to established immunoglobulin ELISAs (3.4% and 2.9% for intra- and inter-assay, respectively)

This methodological approach draws upon established ELISA protocols for protein detection while considering the specific properties of plant tissue extracts and the At2g34840 protein.

What approaches can be used to compare At2g34840 expression across different Arabidopsis tissues?

To systematically compare At2g34840 expression across tissues:

• Tissue collection and processing:

  • Harvest multiple tissue types (leaves, roots, flowers, stems, siliques) at defined developmental stages

  • Flash-freeze samples immediately in liquid nitrogen

  • Process all samples simultaneously using identical extraction protocols to minimize technical variation

• Quantitative Western blotting:

  • Load equal amounts of total protein (30-50 μg) from each tissue

  • Include common reference proteins (actin, tubulin) for normalization

  • Process all tissues on the same gel when possible to minimize inter-gel variation

• ELISA quantification:

  • Develop a quantitative ELISA using purified recombinant At2g34840 protein as standard

  • Prepare standard curves ranging from 0.2-12.5 ng/100 μL

  • Analyze samples in triplicate, calculating both technical and biological variance

• Comparative analysis:

  • Normalize expression data to total protein content or reference proteins

  • Calculate relative expression levels across tissues

  • Perform statistical analysis (ANOVA with post-hoc tests) to identify significant differences

• Validation with orthogonal methods:

  • Correlate protein expression with transcript levels using RT-qPCR

  • Consider immunohistochemistry to visualize tissue-specific localization patterns

This comprehensive approach enables reliable comparison of At2g34840 expression across different Arabidopsis tissues with appropriate statistical validation.

What are common troubleshooting strategies for weak or absent signals when using At2g34840 antibody?

When faced with weak or absent signals, consider these methodological troubleshooting approaches:

• Protein extraction efficiency:

  • Evaluate extraction methods for membrane-associated proteins

  • Add stronger detergents (1-2% SDS) to extraction buffer

  • Sonicate samples to improve protein solubilization

• Antibody binding conditions:

  • Reduce stringency of washing steps (lower salt concentration, fewer washes)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Test higher antibody concentrations (1:500 or 1:250 dilutions)

• Epitope accessibility:

  • For fixed tissues, implement antigen retrieval methods

  • For Western blots, ensure complete protein denaturation with longer boiling time in sample buffer

  • Add reducing agents (β-mercaptoethanol or DTT) to expose epitopes

• Detection sensitivity:

  • Switch to more sensitive detection systems (enhanced chemiluminescence substrates)

  • Implement signal amplification methods (biotin-streptavidin systems)

  • Extend exposure times for Western blots

• Protein degradation assessment:

  • Add additional protease inhibitors to extraction buffers

  • Maintain cold chain throughout sample processing

  • Check for degradation products using total protein stains

Systematically testing these parameters while changing only one variable at a time will help identify the limiting factor in signal detection.

How can At2g34840 antibody be used to study protein-protein interactions in the COPI vesicle system?

For investigating protein-protein interactions involving the Coatomer epsilon subunit:

• Co-immunoprecipitation with mass spectrometry:

  • Use At2g34840 antibody for immunoprecipitation from plant extracts

  • Analyze precipitates by LC-MS/MS to identify interaction partners

  • Compare results between different tissues or treatment conditions

  • Validate key interactions with reverse co-IP experiments

• Proximity labeling approaches:

  • Express BioID or APEX2 fusions of At2g34840 in Arabidopsis

  • Use the antibody to confirm expression and localization of fusion proteins

  • Compare proximity labeling results with traditional co-IP findings

• FRET analysis with immunofluorescence:

  • Perform dual immunostaining with At2g34840 antibody and antibodies against putative interaction partners

  • Use fluorophore-conjugated secondary antibodies suitable for FRET

  • Calculate FRET efficiency to quantify molecular proximity

• Pull-down validation experiments:

  • Express recombinant At2g34840 protein with affinity tags

  • Use the antibody to detect interactions with plant extracts in pull-down assays

  • Confirm specificity with competition experiments using untagged recombinant protein

These methodological approaches can provide complementary evidence for protein-protein interactions involving the Coatomer epsilon subunit in vesicular transport systems.

How does antibody validation for At2g34840 compare with validation standards for other research antibodies?

Antibody validation for At2g34840 should follow rigorous standards comparable to those established for other research antibodies:

• Target specificity validation:

  • The gold standard includes testing in knockout/knockdown lines

  • Recombinant expression systems provide controlled positive controls

  • Mass spectrometry confirmation of immunoprecipitated proteins offers orthogonal validation

• Application-specific validation:

  • For Western blotting: single band of expected molecular weight

  • For immunocytochemistry: localization pattern consistent with known biology

  • For IP applications: enrichment of target protein verified by mass spectrometry

• Cross-reactivity assessment:

  • Testing against related proteins (other coatomer subunits)

  • Evaluation in heterologous expression systems

  • Peptide competition assays to confirm epitope specificity

• Batch-to-batch consistency:

  • Regular quality control testing between lots

  • Consistent performance metrics (titer, specificity, sensitivity)

  • Documentation of validation experiments for reproducibility

• Independent validation:

  • Confirmation of key findings using alternative antibodies or methods

  • Correlation with orthogonal techniques (transcript levels, functional assays)

  • Publication of validation data in peer-reviewed literature

These validation standards align with recent initiatives to improve antibody reliability in research and should be applied rigorously to At2g34840 antibody to ensure experimental reproducibility.

What considerations are important when designing multi-color immunofluorescence experiments with At2g34840 antibody?

For multi-color immunofluorescence experiments:

• Antibody compatibility assessment:

  • At2g34840 antibody is rabbit-derived IgG , requiring careful selection of co-staining antibodies from different host species

  • Test for cross-reactivity between secondary antibodies

  • Perform single-antibody controls to assess bleed-through between channels

• Sequential staining protocols:

  • When using multiple rabbit antibodies, consider sequential staining with complete blocking between rounds

  • Implement fluorophore quenching between sequential staining if necessary

  • Validate that sequential staining doesn't affect epitope recognition

• Spectral considerations:

  • Choose fluorophores with minimal spectral overlap

  • Implement appropriate compensation controls

  • Consider linear unmixing for closely overlapping spectra

• Co-localization analysis:

  • Use appropriate co-localization algorithms (Pearson's, Mander's coefficients)

  • Include appropriate positive and negative co-localization controls

  • Apply statistical analysis to co-localization measurements across multiple cells/samples

• Advanced imaging approaches:

  • Consider super-resolution techniques for resolving closely associated structures

  • Implement deconvolution algorithms to improve signal-to-noise ratio

  • Use spectral imaging for separating closely overlapping fluorophores

These methodological considerations help ensure reliable multi-color imaging results when using At2g34840 antibody in combination with other antibodies for co-localization studies.

How does At2g34840 antibody performance compare to antibodies against other coatomer subunits?

When comparing antibody performance against different coatomer components:

• Target abundance considerations:

  • The epsilon subunit may be expressed at different levels than other coatomer subunits

  • Antibody sensitivity requirements vary according to target abundance

  • Optimization parameters may differ between subunit-specific antibodies

• Epitope accessibility differences:

  • Within the assembled COPI complex, some subunits may have epitopes that are more accessible than others

  • Extraction and denaturation conditions may need optimization for each subunit-specific antibody

  • Native conformation detection capabilities vary between antibodies

• Cross-reactivity profiles:

  • Polyclonal antibodies like At2g34840 antibody may have different cross-reactivity profiles than monoclonal alternatives

  • Epitope conservation across species affects cross-reactivity patterns

  • Validation requirements should be tailored to each antibody's specificity profile

• Application versatility:

  • Compare performance across multiple applications (WB, IP, ICC, ELISA)

  • Document optimal conditions for each application

  • Create standardized protocols that enable cross-comparison between coatomer subunits

A thorough comparative analysis of antibody performance facilitates integrated studies of the entire COPI complex structure and function.

What research questions about vesicle trafficking can be uniquely addressed using At2g34840 antibody?

The At2g34840 antibody enables investigation of several specialized research questions:

• Coatomer complex assembly dynamics:

  • Quantify relative abundance of epsilon subunit during COPI vesicle formation

  • Track temporal recruitment patterns during vesicle budding

  • Compare epsilon subunit incorporation under different cellular conditions

• Plant-specific vesicle trafficking mechanisms:

  • Investigate unique aspects of plant COPI machinery compared to other organisms

  • Study adaptation of vesicle trafficking during plant-specific processes (e.g., cell wall formation, pathogen response)

  • Explore evolutionary conservation of coatomer function across plant species

• Stress response effects on secretory pathway:

  • Monitor changes in At2g34840 expression and localization during abiotic and biotic stress

  • Quantify COPI vesicle formation under stress conditions

  • Correlate secretory pathway alterations with cellular stress responses

• Developmental regulation of vesicular transport:

  • Track At2g34840 expression throughout plant development

  • Correlate coatomer activity with tissue differentiation

  • Identify developmental stages with heightened vesicular transport requirements

These research questions leverage the specificity of At2g34840 antibody to address fundamental aspects of plant vesicle trafficking biology.

What are the comparative advantages of using antibody-based detection versus genetic reporters for studying At2g34840?

Comparing antibody-based detection with genetic reporter approaches reveals distinct advantages:

This comparative analysis helps researchers select the most appropriate approach based on their specific experimental questions and technical constraints.