At2g43600 Antibody

What is the At2g43600 antibody and what organism is it used to study?

At2g43600 antibody is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana At2g43600 protein. It is specifically designed for detecting this target protein in Arabidopsis thaliana (Mouse-ear cress), making it an essential tool for plant molecular biology research . The antibody recognizes the native protein in plant tissues and can be used to investigate protein expression patterns, localization, and function in this model organism. As a research-grade reagent, it is exclusively intended for laboratory research applications and should not be used for diagnostic or therapeutic purposes .

What applications has the At2g43600 antibody been validated for?

The At2g43600 antibody has been validated for specific research applications including ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) techniques . These applications allow researchers to quantify protein expression levels and determine molecular weight and expression patterns, respectively. When designing experiments, it's important to note that each application requires specific optimization of antibody concentration, incubation conditions, and detection methods to ensure optimal signal-to-noise ratio and accurate results. Researchers should follow recommended protocols for each application while incorporating appropriate positive and negative controls to validate findings.

How should the At2g43600 antibody be stored to maintain its activity?

The At2g43600 antibody should be stored at -20°C or -80°C upon receipt to maintain optimal activity and prevent degradation . Repeated freeze-thaw cycles should be avoided as they can denature the antibody and reduce its effectiveness. The antibody is supplied in liquid form in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . This formulation helps maintain stability during storage. For working solutions, aliquoting the antibody into smaller volumes before freezing is recommended to minimize freeze-thaw cycles. For short-term storage (1-2 weeks), the antibody can be kept at 4°C, but long-term storage should always be at -20°C or lower.

How can I determine the optimal working dilution for At2g43600 antibody in Western blotting experiments?

Determining the optimal working dilution for At2g43600 antibody requires systematic titration experiments. Start with a broad range of dilutions (e.g., 1:500, 1:1000, 1:2000, 1:5000) applied to identical protein samples containing your target. The antibody has been affinity-purified , which generally allows for higher dilutions than crude antisera. When analyzing results, look for the dilution that provides the strongest specific signal with minimal background. Often, a titration curve can be plotted to identify the dilution range where signal intensity plateaus. This optimization should be conducted separately for each application (Western blot, ELISA, etc.) and for different sample types (e.g., crude extracts versus purified proteins). Include positive controls (known samples containing At2g43600 protein) and negative controls (samples where the protein is absent or knockout lines) to verify specificity.

What controls should be included when using At2g43600 antibody for immunodetection?

A robust experimental design with appropriate controls is essential for reliable antibody-based research. For At2g43600 antibody experiments, include:

• Positive control: Wild-type Arabidopsis thaliana tissue known to express At2g43600 protein

• Negative control: Ideally, At2g43600 knockout lines or tissues where the protein isn't expressed

• Secondary antibody-only control: Sample incubated with only secondary antibody to identify non-specific binding

• Loading control: Detection of a housekeeping protein (e.g., actin, tubulin) to normalize expression levels

• Pre-adsorption control: Antibody pre-incubated with purified target protein to confirm specificity

Recent advances in antibody validation emphasize the importance of knockout validation, where antibodies are tested against samples from genetic knockout specimens to confirm specificity . This approach is particularly important for polyclonal antibodies like the At2g43600 antibody, which may recognize multiple epitopes on the target protein.

How can I verify the specificity of At2g43600 antibody in my experimental system?

Verifying antibody specificity is crucial for generating reliable scientific data. For At2g43600 antibody, implement a multi-pronged approach:

• Genetic validation: Compare immunodetection signals between wild-type plants and At2g43600 mutant/knockout lines. The signal should be significantly reduced or absent in knockout lines.

• Molecular weight verification: Confirm that the detected protein band in Western blots matches the predicted molecular weight of At2g43600 protein.

• Mass spectrometry validation: Immunoprecipitate proteins using the antibody and analyze by mass spectrometry to confirm capture of the intended target.

• Peptide competition assay: Pre-incubate the antibody with purified recombinant At2g43600 protein or immunogenic peptide before application to your samples. This should dramatically reduce or eliminate specific signal.

• Orthogonal detection methods: Correlate antibody-based detection with other methods such as mRNA expression analysis to confirm expression patterns.

This comprehensive validation approach aligns with recent initiatives to improve research antibody characterization for enhanced reproducibility in scientific research .

What sample preparation techniques optimize At2g43600 protein detection in plant tissues?

Effective sample preparation is critical for successful At2g43600 protein detection in plant tissues. For optimal results:

• Extraction buffer selection: Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail. Phosphatase inhibitors should be added when investigating phosphorylation states.

• Tissue disruption: Flash-freeze tissue in liquid nitrogen and grind to a fine powder before adding extraction buffer to prevent protein degradation.

• Protein solubilization: If At2g43600 is associated with membranes or cell walls, consider sequential extraction with increasing detergent strengths or specialized extraction protocols for membrane-associated proteins.

• Sample clarification: Centrifuge at 10,000-15,000 g for 15 minutes at 4°C to remove insoluble debris before protein quantification.

• Protein quantification: Use Bradford or BCA assay to ensure equal loading across samples.

• Denaturation conditions: For Western blotting, heat samples at 95°C for 5 minutes in Laemmli buffer containing a reducing agent like DTT or β-mercaptoethanol.

For plant cell wall-associated proteins, specialized extraction methods may be necessary, as standard protocols often fail to efficiently extract proteins tightly bound to cell wall components . This is particularly relevant if At2g43600 interacts with cell wall structures or N-acetylglucosamine oligomers in the cell wall matrix.

How can I minimize background and non-specific binding when using At2g43600 antibody?

Achieving clean, specific signals with minimal background is essential for reliable immunodetection. Implement these strategies:

• Blocking optimization: Test different blocking agents (5% non-fat dry milk, 3-5% BSA, commercial blocking reagents) to identify the most effective one for your specific application.

• Antibody dilution: Use the optimal antibody dilution determined through titration experiments. The At2g43600 antibody is antigen-affinity purified , which should help reduce non-specific binding.

• Wash buffer composition: Include 0.05-0.1% Tween-20 in TBS or PBS wash buffers to reduce hydrophobic non-specific interactions.

• Incubation conditions: Perform antibody incubations at 4°C overnight rather than at room temperature to enhance specific binding while reducing non-specific interactions.

• Pre-adsorption: For tissues with high background, consider pre-adsorbing the diluted antibody with an extract from a species that doesn't express At2g43600 to remove antibodies that might cross-react with conserved epitopes.

• Cross-linking fixation: For immunofluorescence applications, optimize fixation protocols as overfixation can lead to epitope masking while underfixation can compromise tissue morphology.

Implementing these methodological refinements should significantly improve signal-to-noise ratio in immunodetection experiments with At2g43600 antibody.

What are the recommended troubleshooting steps when At2g43600 antibody produces weak or no signal?

When confronted with weak or absent signals when using At2g43600 antibody, systematically address potential issues:

• Antibody viability: Check antibody storage conditions—improper storage or multiple freeze-thaw cycles may have compromised activity. The antibody should be stored at -20°C or -80°C with minimal freeze-thaw cycles .

• Protein extraction efficiency: Ensure your extraction protocol efficiently releases At2g43600 protein from plant tissues. If the protein is membrane-associated or in specialized compartments, standard extraction methods may be insufficient.

• Protein degradation: Add fresh protease inhibitors to all buffers and work at 4°C throughout the extraction process to minimize degradation.

• Detection sensitivity: For weak signals, consider using more sensitive detection methods such as chemiluminescence with enhanced substrates or fluorescent secondary antibodies.

• Epitope accessibility: If using fixed tissues, epitope retrieval methods (heat-induced or enzymatic) may help expose antibody binding sites masked during fixation.

• Expression levels: Confirm whether At2g43600 is expressed in your specific tissues or conditions using RT-PCR or RNA-seq data. The protein may be expressed at very low levels requiring enrichment before detection.

• Sample loading: Increase the amount of total protein loaded or consider immunoprecipitation to concentrate the target protein.

• Development time: For Western blots, extend the exposure time during image acquisition to detect weak signals.

By methodically evaluating these factors, researchers can identify and address the specific issues affecting detection of At2g43600 protein.

How can At2g43600 antibody be used to investigate protein-protein interactions in plant stress responses?

At2g43600 antibody can be leveraged for sophisticated protein interaction studies in the context of plant stress responses through several advanced methodologies:

• Co-immunoprecipitation (Co-IP): Use At2g43600 antibody to immunoprecipitate the target protein along with its interaction partners from plant lysates under various stress conditions (e.g., drought, salinity, pathogen infection). Subsequent mass spectrometry analysis can identify novel interaction partners that may be stress-specific .

• Proximity-dependent biotin identification (BioID): Combine the antibody with proximity labeling techniques to capture transient or weak interactions that might be missed by traditional Co-IP approaches.

• Chromatin immunoprecipitation (ChIP): If At2g43600 is involved in transcriptional regulation, ChIP using this antibody can identify genomic regions bound by the protein under different stress conditions.

• Immunofluorescence co-localization: Determine whether At2g43600 protein co-localizes with known stress response proteins using dual immunofluorescence combined with confocal microscopy.

• Förster resonance energy transfer (FRET): Use antibody-based FRET approaches to investigate protein-protein interactions in fixed cells or tissues.

When investigating stress responses, it's essential to carefully control experimental conditions and include appropriate time-course analyses, as protein interactions may be transient or change dynamically during stress response progression . Plant responses to salinity stress are particularly relevant models, as they involve complex transcriptional and post-translational regulation networks.

What strategies can be employed to study post-translational modifications of At2g43600 protein?

Investigating post-translational modifications (PTMs) of At2g43600 protein requires specialized approaches that can be implemented using the antibody:

• Phosphorylation analysis:

  • Immunoprecipitate At2g43600 protein using the antibody, followed by phospho-specific staining or mass spectrometry analysis

  • Use phosphatase treatments of samples before immunoblotting to confirm phosphorylation status

  • Employ Phos-tag SDS-PAGE to separate phosphorylated protein forms before immunodetection

• Glycosylation analysis:

  • Treat samples with specific glycosidases before immunoblotting to identify glycosylated forms

  • Use lectin affinity chromatography in combination with At2g43600 immunodetection

  • This may be particularly relevant if At2g43600 interacts with N-acetylglucosamine oligomers or is involved in cell wall processes

• Ubiquitination detection:

  • Perform immunoprecipitation under denaturing conditions to maintain ubiquitin modifications

  • Use tandem ubiquitin binding entities (TUBEs) to enrich ubiquitinated proteins before immunodetection

  • Probe with both At2g43600 antibody and ubiquitin antibodies to confirm modification

• SUMOylation analysis:

  • Immunoprecipitate with At2g43600 antibody and probe for SUMO proteins

  • Use SUMO-specific proteases to confirm modification

• PTM-specific antibodies:

  • Generate modification-specific antibodies that recognize At2g43600 only when it carries specific PTMs

These approaches should be coupled with appropriate biological treatments that might trigger the PTMs of interest, such as exposure to stress conditions, hormonal treatments, or developmental cues that might regulate At2g43600 function through post-translational mechanisms.

How can At2g43600 antibody be adapted for high-throughput phenotypic screening applications?

Adapting At2g43600 antibody for high-throughput phenotypic screening requires systematic optimization of protocols for increased efficiency while maintaining specificity:

• Microplate-based screening:

  • Optimize At2g43600 antibody for microplate-based ELISA to quantitatively assess protein levels across multiple samples

  • Develop automated liquid handling protocols that minimize antibody consumption while maintaining detection sensitivity

  • Establish robust positive and negative controls for quality control across plates

• Automated immunofluorescence:

  • Adapt At2g43600 antibody protocols for automated immunofluorescence staining in multi-well formats

  • Optimize fixation, permeabilization, and antibody concentration for consistent results across large sample sets

  • Implement automated image acquisition and analysis pipelines to quantify signal intensity, subcellular localization, or co-localization parameters

• Tissue microarrays:

  • Create plant tissue microarrays containing multiple tissue types or treatment conditions

  • Develop standardized immunohistochemistry protocols using At2g43600 antibody for consistent staining across all samples

  • Implement digital pathology approaches for automated scoring and analysis

• Reverse phase protein arrays (RPPA):

  • Adapt At2g43600 antibody for RPPA applications to analyze protein expression across hundreds of samples simultaneously

  • Optimize sample preparation, printing, and detection methods for maximum sensitivity and reproducibility

• Integration with phenotypic data:

  • Correlate At2g43600 immunodetection results with phenotypic measurements to identify associations between protein expression/modification and plant traits

Following the methods used in antibody characterization initiatives like YCharOS , implement standardized validation protocols to ensure reproducibility across high-throughput screening campaigns. This approach is aligned with industry-academic collaborations aimed at improving antibody characterization for research reproducibility.

What methodological considerations are important when analyzing At2g43600 protein in different subcellular compartments?

Analyzing At2g43600 protein in different subcellular compartments requires specialized approaches to preserve compartment integrity and ensure accurate localization:

• Subcellular fractionation:

  • Implement differential centrifugation protocols to separate major cellular compartments (nucleus, cytosol, membranes, organelles)

  • Use density gradient ultracentrifugation for further purification of specific organelles

  • Verify fraction purity using compartment-specific marker proteins

  • Analyze At2g43600 distribution across fractions using the antibody in Western blotting

• Immunofluorescence microscopy:

  • Optimize fixation conditions to preserve cellular architecture while maintaining antibody epitope accessibility

  • Use co-staining with organelle-specific markers to determine precise subcellular localization

  • Implement confocal or super-resolution microscopy for detailed co-localization analysis

  • Consider live-cell imaging approaches using fluorescently-tagged marker proteins combined with fixed-cell immunodetection of At2g43600

• Proximity labeling approaches:

  • Use biotin-based proximity labeling methods (BioID, APEX) to identify proteins in close proximity to At2g43600 in specific compartments

  • Verify proximity labeling results with immunofluorescence co-localization using the antibody

• Immunoelectron microscopy:

  • For ultrastructural localization, adapt At2g43600 antibody for immunogold labeling in transmission electron microscopy

  • Use double-labeling approaches to correlate with known compartment markers

• Cell wall protein analysis:

  • If At2g43600 is associated with cell walls, implement specialized extraction protocols for cell wall-bound proteins

  • Consider enzymatic treatments to release proteins from specific cell wall components

  • This is particularly relevant given potential associations with N-acetylglucosamine oligomers in plant cell walls

These methodological considerations ensure accurate determination of At2g43600 protein localization, which is critical for understanding its function in different cellular contexts and under various experimental conditions.

How can recombinant antibody technology improve At2g43600 protein research?

Recombinant antibody technology offers significant advantages for At2g43600 protein research that can overcome limitations of traditional polyclonal antibodies:

• Enhanced reproducibility:

  • Converting the polyclonal At2g43600 antibody into recombinant formats (single-chain variable fragments, nanobodies) would ensure consistent reagent quality across different production batches

  • The genetic sequence encoding the antibody becomes a permanent resource that can be shared between laboratories, enhancing reproducibility

• Engineered binding properties:

  • Affinity maturation techniques can improve antibody binding strength and specificity

  • Engineering for pH or temperature resistance can expand the range of experimental conditions

  • Humanization or other species adaptations can reduce background in specific applications

• Intracellular expression (intrabodies):

  • Developing At2g43600-specific intrabodies would allow direct functional manipulation in living plants

  • These intrabodies could be designed to block specific protein-protein interactions or interfere with functional domains

• Fusion proteins:

  • Creating recombinant antibody fusions with fluorescent proteins enables real-time tracking of At2g43600 in living cells

  • Fusion with degradation-inducing domains could allow targeted protein knockdown

• Fragment-based approaches:

  • Smaller antibody fragments like nanobodies provide better tissue penetration and access to sterically hindered epitopes

  • These formats are particularly valuable for super-resolution microscopy applications

The transition to renewable recombinant antibody formats represents a significant advancement that would enhance experimental reproducibility while expanding the toolkit available for At2g43600 functional studies .

What considerations are important when integrating At2g43600 antibody data with transcriptomic and proteomic datasets?

Integrating At2g43600 antibody-derived data with -omics datasets requires careful methodological considerations:

• Correlation analysis:

  • Design experiments to collect both protein (using At2g43600 antibody) and mRNA expression data from the same samples

  • Implement statistical approaches to correlate protein abundance with transcript levels to identify post-transcriptional regulation

  • Collect data across multiple conditions or time points to capture dynamic regulation

• Data normalization:

  • Develop robust normalization strategies to compare antibody-based quantification with mass spectrometry-based proteomics data

  • Use internal controls consistently across platforms to enable accurate integration

• Temporal considerations:

  • Account for time lags between transcriptional and translational responses when interpreting integrated datasets

  • Design time-course experiments with appropriate sampling intervals to capture both rapid transcriptional changes and slower protein abundance changes

• Spatial resolution:

  • Compare cell type-specific transcriptomics with immunolocalization data to understand tissue-specific regulation

  • Cell-type-specific transcriptional responses may reveal functional contexts for At2g43600 protein

• Functional networks:

  • Use protein interaction data (obtained via Co-IP with At2g43600 antibody) to build functional networks

  • Overlay transcriptional co-expression data to identify modules of co-regulated genes and proteins

  • Implement machine learning approaches to predict functional relationships from integrated datasets

• Data visualization:

  • Develop visualization tools that simultaneously represent protein abundance, localization, modification state, and transcript levels

This multi-dimensional approach provides a comprehensive understanding of At2g43600 regulation and function within the cellular context that single-platform analyses cannot achieve.

How can the specificity of At2g43600 antibody be verified for studying protein in different plant species?

When extending At2g43600 antibody use to other plant species, rigorous cross-reactivity validation is essential:

• Sequence homology analysis:

  • Perform bioinformatic analysis to identify homologs of At2g43600 in target species

  • Assess sequence conservation in epitope regions to predict potential cross-reactivity

  • Generate sequence alignments highlighting conserved and variable regions across species

• Western blot validation:

  • Perform side-by-side Western blots with protein extracts from Arabidopsis and target species

  • Compare band patterns, molecular weights, and signal intensities

  • Include multiple tissue types to account for differential expression patterns

• Immunoprecipitation-mass spectrometry:

  • Use the antibody for immunoprecipitation from target species extracts

  • Confirm target identity by mass spectrometry analysis

  • Identify any off-target proteins that might be recognized in the new species

• Genetic validation:

  • Test antibody reactivity in knockout/knockdown lines of the homologous gene in the target species

  • Compare wild-type and mutant signals to confirm specificity

• Pre-adsorption controls:

  • Pre-incubate antibody with recombinant Arabidopsis At2g43600 protein before applying to target species samples

  • Loss of signal indicates shared epitopes between species

• Epitope mapping:

  • Identify the specific epitopes recognized by the antibody

  • Synthesize peptides representing these epitopes from both Arabidopsis and target species

  • Compare antibody binding to these peptides to assess cross-reactivity at the epitope level