At2g29930 Antibody

What is At2g29930 and why is it studied in Arabidopsis thaliana?

At2g29930 refers to a specific gene locus in Arabidopsis thaliana (mouse-ear cress), a model plant organism widely used in molecular biology and genetics research. The protein encoded by this gene is studied to understand various cellular processes in plants. Antibodies against this protein allow researchers to detect, quantify, and localize it within plant tissues or cell extracts. Research using At2g29930 antibodies typically focuses on protein expression patterns, protein-protein interactions, and functional analyses in developmental or stress-response studies. When designing experiments with this antibody, researchers should consider appropriate controls, including wild-type versus knockout comparisons and validation in multiple experimental systems .

What applications are validated for At2g29930 antibody?

At2g29930 antibody has been validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) applications. These techniques allow researchers to detect and quantify the target protein in complex biological samples. For Western blotting, the antibody enables identification of the specific protein band among numerous proteins separated by gel electrophoresis. For ELISA, the antibody can be used in various formats, including direct, indirect, sandwich, or competitive assays, depending on the experimental requirements. Researchers should optimize antibody concentration, incubation time, and washing steps for each specific application to maximize signal-to-noise ratio .

How should I store and handle At2g29930 antibody to maintain its activity?

Proper storage and handling of At2g29930 antibody is crucial for maintaining its activity and specificity. Upon receipt, the antibody should be stored at -20°C or -80°C, and repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of binding activity. The antibody is supplied in a liquid form, preserved in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4. This formulation helps maintain antibody stability during storage. When working with the antibody, aliquot into smaller volumes for single use to prevent contamination and repeated freeze-thaw cycles. Always handle the antibody using clean technique and appropriate laboratory safety measures .

What controls should I include when using At2g29930 antibody in my experiments?

When designing experiments with At2g29930 antibody, several controls should be incorporated to ensure reliable and interpretable results:

• Positive control: Include a sample known to express At2g29930 protein, such as wild-type Arabidopsis thaliana tissue.

• Negative control: Use samples from At2g29930 knockout plants or tissues where the protein is not expressed.

• Primary antibody control: Perform parallel experiments without the primary antibody to assess non-specific binding of the secondary detection system.

• Isotype control: Include a non-specific IgG from the same species (rabbit) to evaluate background signal.

• Blocking peptide control: When available, use the immunogen peptide to competitively inhibit specific antibody binding.

These controls help distinguish specific signal from background and validate the specificity of the observed results, which is especially important given the polyclonal nature of this antibody .

How can I optimize At2g29930 antibody concentration for different experimental systems?

Optimization of At2g29930 antibody concentration is essential for achieving high signal-to-noise ratios in your experiments. Based on recent findings in antibody titration studies, most antibodies reach their saturation plateau at concentrations between 0.62 and 2.5 μg/mL . For At2g29930 antibody specifically, I recommend the following titration approach:

• Perform a serial dilution experiment starting at 2.5 μg/mL and diluting fourfold down to 0.039 μg/mL.

• Evaluate both signal intensity and background for each concentration.

• Select the lowest concentration that provides robust and specific signal.

For Western blotting, consider testing multiple blocking reagents (BSA, milk, commercial blockers) as different blockers can affect antibody performance. For ELISA, optimize both coating and detection antibody concentrations independently. Research shows that antibodies used at concentrations above 2.5 μg/mL often show limited response to further titration and may contribute to higher background .

What strategies can resolve specificity issues with At2g29930 antibody in complex plant extracts?

Resolving specificity issues with At2g29930 antibody in complex plant extracts requires a multi-faceted approach:

• Sample preparation optimization: Modify extraction buffers to reduce non-specific binding. Consider including detergents like 0.1% Triton X-100 or varying salt concentrations to reduce non-specific interactions.

• Pre-absorption techniques: Incubate the antibody with extracts from plants lacking At2g29930 expression to remove antibodies that bind non-specifically.

• Affinity purification: Since this antibody is already antigen-affinity purified , you may need to perform additional immunodepletion against cross-reactive antigens.

• Signal verification: Confirm results using alternative detection methods or a second antibody targeting a different epitope of At2g29930.

• Background reduction: As recent research shows, high antibody concentrations (above 2.5 μg/mL) often lead to increased background with minimal gains in specific signal . Consider reducing antibody concentration if background is problematic.

• Technical controls: Include proper negative controls like knockout or knockdown lines to distinguish true signal from background.

Implementing these strategies systematically can help isolate specific At2g29930 signals from complex plant extracts.

How can I apply At2g29930 antibody in multi-protein detection systems?

Incorporating At2g29930 antibody into multi-protein detection systems requires careful planning to avoid cross-reactivity and ensure optimal signal for each target. Consider these methodological approaches:

• Sequential detection: For Western blots, strip and reprobe membranes sequentially, starting with the lowest abundance target (potentially At2g29930) and moving to more abundant proteins.

• Multiplexed fluorescence detection: Utilize antibodies raised in different host species and corresponding species-specific secondary antibodies conjugated to different fluorophores.

• Optimization of staining volumes: Recent research indicates that reducing staining volume can affect signal intensity, particularly for antibodies used at low concentrations targeting highly expressed epitopes . For At2g29930 detection within a multiplexed panel, consider using:

  • Standard volume (50 μL) when targeting low-abundance epitopes

  • Reduced volume (25 μL) for medium to high-abundance targets to optimize signal-to-background ratio

• Cell count considerations: When working with isolated plant cells, adjust the cell count based on experimental needs. Research shows that reducing cell numbers during staining can counteract negative effects of reduced staining volumes .

• Antibody panel design: If designing a comprehensive protein detection panel including At2g29930, consider that total background increases with the number of antibodies in the panel, particularly those used at high concentrations .

For multiplexed immunofluorescence applications, careful validation of each antibody in the panel is essential to ensure specificity and minimal cross-reactivity.

What approaches can improve At2g29930 antibody sensitivity for detecting low-abundance proteins?

Detecting low-abundance At2g29930 protein requires enhancing antibody sensitivity through several advanced techniques:

• Signal amplification systems:

  • Utilize tyramide signal amplification (TSA) which can increase sensitivity 10-100 fold

  • Consider biotin-streptavidin amplification systems for enhanced detection

  • Employ polymer-based detection systems that carry multiple enzyme molecules per antibody binding event

• Sample enrichment:

  • Use immunoprecipitation to concentrate the target protein before detection

  • Apply subcellular fractionation to reduce sample complexity

  • Consider protein extraction methods optimized for membrane or nuclear proteins depending on At2g29930 localization

• Optimized antibody concentration:

  • Recent research indicates that antibodies at lower concentrations (0.62 μg/mL and below) show near-linear response to dilution , suggesting proper titration is critical

  • Use the minimum effective antibody concentration to reduce background

• Enhanced imaging/detection:

  • For immunofluorescence, use high-sensitivity cameras and appropriate exposure settings

  • For Western blotting, consider longer exposure times with low-fluorescence membranes

  • Employ digital image analysis software for quantification of weak signals

• Reducing background:

  • Optimize blocking solutions specifically for plant extracts

  • Consider extended washing steps to remove non-specific binding

  • Use detergents appropriate for maintaining At2g29930 antigenicity while reducing background

When implementing these approaches, always include appropriate controls to validate that the enhanced signal represents true At2g29930 detection rather than amplified background.

What are the most common causes of false negative results with At2g29930 antibody?

False negative results when using At2g29930 antibody can occur for multiple reasons, each requiring specific troubleshooting approaches:

• Protein denaturation: As the At2g29930 antibody was raised against recombinant protein , it may recognize conformational epitopes. Harsh extraction conditions or sample preparation methods may denature the protein, preventing antibody recognition. Try milder extraction protocols or native conditions where possible.

• Epitope masking: Post-translational modifications or protein-protein interactions may mask the epitope. Consider using alternative extraction buffers or denaturing conditions to expose masked epitopes.

• Insufficient antibody concentration: While excessive antibody can increase background, insufficient concentrations may result in no signal. Titrate the antibody carefully, noting that the optimal concentration for detection will depend on target abundance.

• Degraded antibody: The recommended storage is at -20°C or -80°C, avoiding repeated freeze-thaw cycles . Degraded antibody will lose binding capacity. Aliquot the antibody upon receipt to minimize freeze-thaw cycles.

• Low protein expression: At2g29930 may be expressed at levels below detection limits. Consider enrichment techniques or more sensitive detection systems.

• Inappropriate secondary antibody: Ensure you're using a secondary antibody that specifically recognizes rabbit IgG (as this is a rabbit polyclonal antibody ).

• Incompatible buffer systems: The preservation buffer (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) may interact with certain experimental buffers. Ensure compatibility between antibody buffer and experimental conditions.

If experiencing false negatives, systematically address each of these potential issues while including appropriate positive controls.

How can I distinguish between specific binding and background when using At2g29930 antibody?

Distinguishing between specific binding and background signals is critical for accurate interpretation of results with At2g29930 antibody. Implement these methodological approaches:

• Knockout/knockdown controls: The most definitive approach is comparing wild-type samples with those where At2g29930 has been genetically removed or suppressed. Any signal remaining in knockout samples represents non-specific binding.

• Signal pattern analysis: Specific binding should correlate with known or predicted expression patterns and cellular localization of At2g29930. Signals appearing uniformly across all tissues or cellular compartments likely represent background.

• Titration response assessment: Recent research shows that specific antibody signals demonstrate a dose-dependent response to titration, particularly at concentrations between 0.62 and 2.5 μg/mL . Background signals often don't show this pattern of response.

• Competitive binding experiments: Pre-incubating the antibody with purified At2g29930 protein or the immunogen peptide should diminish specific signals but not affect background.

• Sequential probing approach: When performing multiplex detection, sequence the order of antibody application from lowest to highest abundance targets to minimize cross-reactivity .

• Background quantification in negative controls: Quantify signal in negative controls to establish a baseline threshold above which signals can be considered specific. Statistical approaches like signal-to-noise ratio calculation can provide objective criteria.

• Alternative detection methods: Validate observed signals using an independent detection method or a second antibody recognizing a different epitope of At2g29930.

By implementing these approaches systematically, you can establish confidence in distinguishing specific At2g29930 signals from experimental background.

What methodological approaches can resolve cross-reactivity issues with At2g29930 antibody?

Cross-reactivity with non-target proteins is a common challenge with polyclonal antibodies like the At2g29930 antibody . These methodological approaches can help resolve such issues:

• Antibody pre-absorption:

  • Incubate the antibody with tissue lysates from At2g29930 knockout plants

  • Remove the bound antibodies using protein A/G beads

  • Use the remaining unbound antibody fraction for your experiments

• Cross-reactive protein identification:

  • Perform immunoprecipitation followed by mass spectrometry to identify cross-reactive proteins

  • Use bioinformatics to analyze sequence similarities between At2g29930 and potential cross-reactive proteins

  • Design blocking strategies based on identified cross-reactive epitopes

• Epitope-specific elution:

  • Use the immunogen peptide to specifically elute only those antibodies that bind to the intended epitope

  • This enriches for antibodies with the desired specificity

• Differential detection strategies:

  • Incorporate multiple antibodies targeting different epitopes of At2g29930

  • True signals should show correlation across different antibodies

• Validation in multiple systems:

  • Recent advances in antibody specificity testing emphasize multi-system validation

  • Test the antibody in different contexts: wild-type vs. knockout, overexpression systems, different tissue types

• Optimized immunoblotting conditions:

  • Adjust blocking reagents (try BSA, milk, commercial blockers)

  • Vary detergent concentrations in washing steps

  • Optimize incubation times to favor high-affinity specific binding over lower-affinity cross-reactive binding

• Consideration of alternative detection methods:

  • If cross-reactivity persists, consider alternative approaches like RNA detection or tagged protein expression

Recent research on antibody design emphasizes that high specificity can be engineered to distinguish between closely related protein subtypes or mutants , suggesting that more specific antibodies may be available in the future.

How can At2g29930 antibody be adapted for single-cell analysis techniques?

Adapting At2g29930 antibody for single-cell analysis requires optimization for enhanced sensitivity and specificity at the individual cell level:

• Antibody conjugation strategies:

  • Direct conjugation with fluorophores or oligos for CITE-seq applications

  • When preparing oligo-conjugated At2g29930 antibody, optimal concentration appears to be between 0.62-2.5 μg/mL, as research shows antibodies in this range provide optimal signal-to-background ratio

  • Higher concentrations (above 2.5 μg/mL) contribute disproportionately to background in single-cell analyses without significant gains in specific signal

• Staining protocol optimization:

  • Reduce staining volume (25 μL vs. 50 μL) when working with smaller cell numbers

  • For plant single-cell applications, protoplast preparation must preserve epitope integrity

  • Consider reducing cell counts during staining to 0.2×10^6 cells for optimal signal detection

• Background reduction strategies:

  • Recent single-cell antibody research reveals that background signal in empty droplets can constitute a major fraction of sequencing reads

  • Pre-sort cells to remove debris that may bind antibodies non-specifically

  • Implement computational approaches to distinguish signal from background in single-cell data

• Multiplexed detection considerations:

  • When incorporating At2g29930 antibody into multiplexed panels, adjust its concentration based on epitope abundance

  • Consider the position effect in antibody panels, as certain antibodies may interfere with others in multiplexed settings

• Validation in single-cell context:

  • Confirm antibody performance in bulk samples before transitioning to single-cell applications

  • Evaluate antibody compatibility with single-cell fixation and permeabilization protocols

Optimizing these parameters will allow successful adaptation of At2g29930 antibody for demanding single-cell applications in plant research.

What are the considerations for using At2g29930 antibody in combination with emerging antibody design technologies?

The integration of At2g29930 antibody with emerging antibody technologies presents several advanced research opportunities:

• Computational antibody design integration:

  • Recent breakthroughs in de novo antibody design demonstrate that precision design can achieve high specificity without prior antibody information

  • Consider using computational approaches to design complementary antibodies targeting alternative At2g29930 epitopes for validation

  • Newly designed antibodies could be engineered for specific functions (blocking protein interactions, detecting specific conformations)

• Structural considerations:

  • Modern antibody design utilizes atomic-level physicochemical interactions

  • Analyzing the predicted structure of At2g29930 could inform better epitope selection

  • For proteins like At2g29930 without experimentally resolved structures, computational prediction can still enable successful antibody design

• Specificity engineering:

  • Current At2g29930 antibody may have limitations in distinguishing closely related proteins

  • New technologies can design antibodies with enhanced specificity for distinguishing protein variants with only few amino acid differences

  • Consider computational redesign of current At2g29930 antibody for improved specificity

• Format adaptation considerations:

  • Current At2g29930 antibody is available as a full IgG

  • Consider engineering alternative formats (Fab, scFv, nanobody) for specific applications

  • Different formats may provide advantages for certain applications like intracellular targeting or tissue penetration

• Production system optimization:

  • Modern antibody engineering considers developability characteristics early in design

  • Optimized antibodies show improved stability, reduced aggregation, and higher expression yields

  • Consider production system compatibility when designing new At2g29930 antibody variants

By integrating current At2g29930 antibody research with emerging computational design approaches, researchers can develop enhanced reagents with superior specificity and performance characteristics.

How can At2g29930 antibody performance be validated for reproducible research?

Ensuring reproducible results with At2g29930 antibody requires comprehensive validation across multiple dimensions:

• Multi-batch comparison:

  • Test multiple production lots of the antibody under identical conditions

  • Quantify batch-to-batch variability in signal intensity, background, and specificity

  • Establish acceptance criteria for new antibody batches based on performance metrics

• Cross-laboratory validation protocol:

  • Develop a standardized protocol that can be shared between research groups

  • Include detailed methods for sample preparation, antibody dilution, incubation conditions, and data analysis

  • Share positive control samples that can be used to calibrate results between laboratories

• Comprehensive documentation:

  • Record detailed antibody information including:

    • Catalog number (CSB-PA801031XA01DOA)

    • Lot number

    • Immunogen sequence (Recombinant Arabidopsis thaliana At2g29930 protein)

    • Validation methods performed

    • Known limitations and cross-reactivities

• Quantitative performance metrics:

  • Establish quantitative criteria for antibody performance:

    • Signal-to-noise ratio in relevant applications

    • Detection limit under standardized conditions

    • Linear range of detection

    • Reproducibility metrics (coefficient of variation)

• Orthogonal validation approaches:

  • Confirm key results using alternative methods not dependent on the antibody

  • Correlate protein detection with mRNA expression data

  • Validate subcellular localization using tagged protein expression

• Antibody authentication:

  • Confirm antibody identity via mass spectrometry

  • Verify species reactivity claims (specifically Arabidopsis thaliana)

  • Test cross-reactivity with related plant species

• Digital validation record:

  • Create a digital record of validation experiments accessible to the scientific community

  • Include raw data, experimental protocols, and analysis methods

  • Update the record as new validation information becomes available

Implementing these validation practices will enhance research reproducibility and build confidence in results obtained using At2g29930 antibody.

What are the recommended best practices for experimental design using At2g29930 antibody?

Based on current research and antibody characteristics, these best practices will optimize experimental outcomes when using At2g29930 antibody:

• Comprehensive experimental planning:

  • Design experiments with appropriate positive and negative controls

  • Include At2g29930 knockout/knockdown samples when possible

  • Plan for biological and technical replicates to ensure statistical validity

• Antibody handling and storage:

  • Store at recommended temperature (-20°C or -80°C)

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Maintain proper documentation of antibody lot numbers and performance characteristics

• Application-specific optimization:

  • For Western blotting: Determine optimal concentration through titration, starting at 1:1000 dilution

  • For ELISA: Establish standard curves using purified recombinant At2g29930 protein

  • For each application, optimize blocking reagents, incubation times, and washing procedures

• Context-appropriate concentration selection:

  • Use concentrations between 0.62-2.5 μg/mL for optimal signal-to-background ratio

  • Consider further dilution for highly expressed targets to maintain linear detection range

  • Avoid concentrations above 2.5 μg/mL which contribute minimally to specific signal but increase background

• Validation in relevant systems:

  • Test antibody performance in the specific plant tissues/cells relevant to your research

  • Validate antibody in both wild-type and manipulated (overexpression/knockout) systems

  • Consider developmental stage and environmental conditions when interpreting results

• Transparent reporting:

  • Document all experimental conditions in publications

  • Report antibody details including catalog number, lot, dilution, and incubation conditions

  • Share validation data as supplementary material in publications

• Continuous performance monitoring:

  • Maintain reference samples to monitor antibody performance over time

  • Re-validate new antibody lots against established standards

  • Document any changes in antibody performance characteristics