At3g55900 Antibody

What is the At3g55900 protein and why is it studied?

The At3g55900 gene in Arabidopsis thaliana encodes a protein with important functional roles in plant cellular processes. This protein (Uniprot ID: Q6AWW4) has gained research interest due to its involvement in plant developmental pathways and stress responses. The antibody targeting this protein enables researchers to investigate its expression patterns, localization, and potential interactions with other proteins in various experimental conditions. While specific functional characterization studies are ongoing, the antibody serves as a crucial tool for elucidating the protein's role in plant biology .

What are the technical specifications of the At3g55900 antibody?

The At3g55900 antibody (Product Code: CSB-PA721450XA01DOA) is a polyclonal antibody raised in rabbits using recombinant Arabidopsis thaliana At3g55900 protein as the immunogen. It is supplied in liquid form, containing 50% glycerol with 0.01M PBS (pH 7.4) and 0.03% Proclin 300 as a preservative. The antibody is purified using antigen affinity purification methods to enhance specificity. For storage, it should be kept at -20°C or -80°C to maintain stability, and repeated freeze-thaw cycles should be avoided to preserve antibody functionality .

What applications is the At3g55900 antibody validated for?

The At3g55900 antibody has been validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) applications. When using this antibody for Western blotting, it's essential to optimize experimental conditions including sample preparation, protein loading, blocking conditions, antibody concentration, and incubation times. As with many plant protein antibodies, cross-validation of results using complementary techniques is recommended to ensure specificity and accuracy in identifying the target antigen .

How should I optimize Western blotting protocols for the At3g55900 antibody?

For optimal Western blotting results with the At3g55900 antibody, a methodical optimization approach is recommended:

• Sample preparation: Extract proteins from Arabidopsis tissues using an appropriate buffer system (e.g., RIPA buffer with protease inhibitors). Consider using different extraction methods for membrane-associated proteins if standard protocols yield poor results.

• Antibody dilution: Start with a 1:1000 dilution and adjust based on signal strength. A titration series (1:500, 1:1000, 1:2000) can help determine optimal concentration.

• Blocking conditions: Test both 5% non-fat dry milk and 5% BSA in TBST as blocking agents, as plant antibodies may perform differently with various blocking solutions.

• Incubation conditions: Incubate primary antibody overnight at 4°C for optimal binding, followed by appropriate HRP-conjugated secondary antibody (anti-rabbit IgG) at 1:5000 to 1:10000 dilution.

• Controls: Include both positive controls (known At3g55900-expressing tissues) and negative controls (tissues with low/no expression) to validate specificity.

This optimization approach follows standard protocols similar to those used with other plant protein antibodies in immunoblotting applications .

What are the recommended ELISA protocols for At3g55900 antibody?

When designing ELISA experiments with the At3g55900 antibody, consider the following protocol framework:

Indirect ELISA Protocol:

• Coat microplate wells with target protein or plant extract (typically 1-10 μg/ml) in coating buffer (carbonate-bicarbonate, pH 9.6) overnight at 4°C

• Wash with PBS-T (PBS + 0.05% Tween-20) three times

• Block with 2-5% BSA in PBS-T for 1-2 hours at room temperature

• Apply At3g55900 antibody at optimized dilution (start with 1:500 to 1:2000) in blocking buffer for 1-2 hours at room temperature

• Wash five times with PBS-T

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

• Wash five times with PBS-T

• Develop with TMB substrate and measure absorbance at 450 nm

Optimization Table for ELISA Conditions:

This approach is based on standard ELISA methodology used for plant antibodies and should be optimized specifically for the At3g55900 antibody .

How can I validate the specificity of the At3g55900 antibody?

Validating antibody specificity is crucial for reliable research outcomes. For the At3g55900 antibody, implement the following multi-approach validation strategy:

• Knockout/knockdown controls: Test the antibody in Arabidopsis At3g55900 mutant or knockdown lines, where signal reduction/absence confirms specificity.

• Immunoprecipitation followed by mass spectrometry: Perform IP with the antibody and analyze pulled-down proteins by mass spectrometry to confirm target protein identity.

• Pre-adsorption test: Pre-incubate the antibody with excess purified recombinant At3g55900 protein before application in Western blot or ELISA; signal reduction indicates specificity.

• Cross-reactivity assessment: Test the antibody against related Arabidopsis proteins to evaluate potential cross-reactivity.

• Correlation with transcript levels: Compare protein detection with RT-qPCR measurements of At3g55900 transcript levels across different tissues or conditions.

These validation approaches are consistent with established antibody validation methodologies used in plant biology research and will help ensure experimental reliability .

Why might I observe weak or no signal when using the At3g55900 antibody?

Several factors may contribute to weak or absent signals when working with the At3g55900 antibody:

• Protein expression levels: At3g55900 may be expressed at low levels in your samples. Consider using tissues or conditions where expression is known to be higher, or concentrate proteins during sample preparation.

• Extraction efficiency: The protein may require specialized extraction methods if it is membrane-associated or part of protein complexes. Try different extraction buffers with varying detergent concentrations.

• Epitope masking: Post-translational modifications or protein interactions may mask the epitope. Test denaturing conditions or different sample preparation methods.

• Antibody degradation: Repeated freeze-thaw cycles can reduce antibody activity. Aliquot the antibody upon receipt and avoid multiple freeze-thaw cycles.

• Detection sensitivity: For Western blots, consider using more sensitive detection methods like enhanced chemiluminescence (ECL) substrates or fluorescent secondary antibodies.

A systematic approach to troubleshooting these factors is recommended, changing one variable at a time and documenting results to identify the specific issue .

How can I reduce background when using the At3g55900 antibody?

Excessive background is a common challenge when working with plant antibodies. For the At3g55900 antibody, implement these strategies to improve signal-to-noise ratio:

• Optimize blocking: Test different blocking agents (BSA, non-fat dry milk, commercial blocking buffers) at various concentrations (2-5%) and times (1-2 hours).

• Increase washing stringency: Increase the number of washes (5-6 times instead of 3) and duration (10 minutes per wash) using TBST or PBST with slightly increased Tween-20 concentration (0.1% instead of 0.05%).

• Dilute antibody appropriately: Excessive antibody concentration can increase background. Try more dilute antibody solutions while extending incubation time.

• Add blocking agents to antibody diluent: Include 0.1-0.5% blocking agent in the antibody dilution buffer to reduce non-specific binding.

• Pre-adsorb secondary antibody: If using plant tissues, pre-adsorb secondary antibodies with plant extract powder to reduce cross-reactivity with plant proteins.

These approaches have proven effective for reducing background with plant antibodies in various immunoassay formats .

What are the best positive controls for validating At3g55900 antibody performance?

Selecting appropriate positive controls is essential for validating antibody performance. For the At3g55900 antibody, consider these control options:

• Recombinant At3g55900 protein: Using purified recombinant protein as a positive control provides the most direct validation of antibody binding.

• Overexpression systems: Arabidopsis plants or cell cultures overexpressing the At3g55900 gene (with or without epitope tags) can serve as strong positive controls.

• Developmental stage-specific samples: If expression data for At3g55900 is available, select tissues or developmental stages known to have higher expression levels.

• Stress-induced samples: If At3g55900 is responsive to particular stresses, treated samples may provide enhanced expression for antibody validation.

• Cross-species validation: For conserved proteins, consider testing the antibody in closely related plant species where homologs may be more abundantly expressed.

When using these controls, include a loading control antibody (such as anti-actin or anti-tubulin) to normalize for protein loading variations .

How can I use the At3g55900 antibody for co-immunoprecipitation (Co-IP) studies?

Although the At3g55900 antibody is primarily validated for ELISA and Western blot applications, it can potentially be adapted for co-immunoprecipitation studies to investigate protein-protein interactions. Consider this methodological approach:

• Buffer optimization: Use gentle lysis buffers (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease inhibitors) that preserve protein-protein interactions.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody coupling: For better results, consider covalently coupling the At3g55900 antibody to protein A/G beads using cross-linking reagents like dimethyl pimelimidate (DMP).

• Controls: Include IgG-only controls and, if possible, samples from At3g55900 knockout/knockdown plants.

• Gentle elution: Use non-denaturing elution methods first to preserve interacting proteins, followed by more stringent elution for the primary target.

• Validation: Confirm pulled-down proteins by Western blotting and/or mass spectrometry.

This approach adapts standard Co-IP protocols for plant research applications while considering the specific characteristics of plant cellular components .

Can the At3g55900 antibody be used for immunohistochemistry or immunofluorescence?

While the At3g55900 antibody is not explicitly validated for immunohistochemistry (IHC) or immunofluorescence (IF), researchers may adapt it for these applications using the following protocol considerations:

• Fixation optimization: Test different fixatives (4% paraformaldehyde, ethanol-acetic acid, etc.) as plant tissues often require specific fixation protocols.

• Antigen retrieval: Incorporate heat-induced or enzymatic antigen retrieval steps to expose epitopes that may be masked during fixation.

• Permeabilization: Optimize cell wall and membrane permeabilization using detergents (0.1-0.5% Triton X-100) or enzymatic digestion.

• Blocking: Use 3-5% BSA with 0.3% Triton X-100 in PBS, potentially adding 5-10% normal serum matching the secondary antibody host.

• Antibody dilution: Start with higher concentrations (1:100 to 1:250) than used for Western blotting.

• Controls: Include peptide competition controls and tissues from knockout/knockdown plants.

• Detection system: Test both conventional fluorophore-conjugated secondary antibodies and signal amplification systems (e.g., tyramide signal amplification) for optimal sensitivity.

Success in adapting antibodies for these applications varies, and extensive optimization may be required .

What approaches can be used to quantify At3g55900 protein expression levels?

For quantitative analysis of At3g55900 protein expression across different conditions or tissues, consider these methodological approaches:

• Quantitative Western blotting:

  • Use gradient-loaded standard curves of recombinant At3g55900 protein

  • Employ fluorescent secondary antibodies for wider linear detection range

  • Include consistent loading controls (actin, tubulin, or total protein stains like Ponceau S)

  • Analyze band intensity using software like ImageJ with appropriate background correction

• Quantitative ELISA:

  • Develop a sandwich ELISA using the At3g55900 antibody paired with another antibody recognizing a different epitope

  • Create standard curves using purified recombinant protein

  • Optimize sample dilutions to ensure measurements fall within the linear range

• Proteomics approaches:

  • Use SILAC or TMT labeling combined with immunoprecipitation for relative quantification

  • Consider selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) for targeted quantification

• Normalization strategies:

  • Express results relative to total protein content

  • Use multiple reference proteins for more reliable normalization

  • Consider tissue-specific reference proteins based on expression stability

These quantitative approaches should be validated for the specific experimental context to ensure accurate measurement of At3g55900 protein levels .

How does the performance of polyclonal vs. monoclonal antibodies compare for plant proteins like At3g55900?

When considering antibody options for plant protein research, understanding the comparative advantages of polyclonal versus monoclonal antibodies is essential:

Polyclonal Antibodies (like the At3g55900 antibody):

• Recognize multiple epitopes on the target protein, potentially increasing detection sensitivity

• More tolerant of minor protein denaturation or modifications

• Generally less expensive and faster to produce

• May show batch-to-batch variation requiring re-optimization

• Potential for higher cross-reactivity with related proteins

Monoclonal Antibodies:

• Recognize a single epitope with high specificity

• Provide consistent performance across batches

• May have lower sensitivity for certain applications

• Production is more time-consuming and expensive

• May be more sensitive to epitope loss through denaturation or modification

For Arabidopsis proteins like At3g55900, polyclonal antibodies often provide a good starting point for research due to their higher sensitivity, while monoclonal antibodies may be developed later for more standardized applications requiring higher specificity .

What are the best practices for antibody validation in plant research compared to other systems?

Antibody validation in plant research presents unique challenges compared to mammalian systems. Based on comparative methodologies, these best practices are recommended for the At3g55900 antibody:

• Genetic knockout validation: Use T-DNA insertion lines or CRISPR/Cas9-generated knockouts of At3g55900 as negative controls - this is the gold standard for plant antibody validation.

• Recombinant expression: Express tagged versions of At3g55900 in heterologous systems or Arabidopsis for positive controls.

• Orthogonal detection methods: Correlate antibody results with RNA-seq or RT-qPCR data across tissues or conditions.

• Independent antibody verification: When possible, compare results using antibodies raised against different epitopes of the same protein.

• Cross-species validation: Test specificity across related plant species with varying degrees of protein conservation.

• Publication of validation data: Document and publish complete validation data including blot images showing both positive and negative controls.

The plant research community has increasingly recognized the importance of rigorous antibody validation to improve experimental reproducibility, though standardized validation criteria specifically for plant antibodies are still evolving .

How can I integrate At3g55900 antibody data with other omics approaches in Arabidopsis research?

Integrating antibody-based protein detection with other omics datasets provides a more comprehensive understanding of At3g55900 function. Consider these methodological approaches:

• Transcriptomics integration:

  • Compare protein levels detected by the At3g55900 antibody with transcript levels across tissues or treatments

  • Identify conditions where protein and mRNA levels diverge, suggesting post-transcriptional regulation

• Proteomics correlation:

  • Validate mass spectrometry-based proteomics data with targeted antibody detection

  • Use antibody-based enrichment (immunoprecipitation) followed by mass spectrometry to identify interacting partners

• Metabolomics connections:

  • Correlate At3g55900 protein levels with metabolite profiles to identify potential functional relationships

  • In mutant/overexpression lines, connect altered protein levels with metabolic changes

• Phenomics annotation:

  • Link protein expression patterns with phenotypic data from different growth conditions

  • Document protein localization in relation to developmental or stress-response phenotypes

• Data visualization and integration:

  • Use tools like Araport, BAR, or custom R/Python scripts to visualize antibody-derived data alongside other omics datasets

  • Develop network models incorporating protein expression data with interaction and functional genomics data

This multi-omics integration approach enhances the value of antibody-based detection by placing it within a broader systems biology context .

What are the emerging applications for plant protein antibodies in Arabidopsis research?

Antibodies against plant proteins like At3g55900 are finding expanding applications in cutting-edge research areas:

• Single-cell proteomics: Adapting antibodies for use in single-cell protein profiling to understand cellular heterogeneity within plant tissues.

• Super-resolution microscopy: Optimizing antibodies for techniques like STORM or PALM to visualize protein localization at nanometer resolution.

• Protein interaction dynamics: Using antibodies in combination with proximity labeling approaches (BioID, APEX) to capture dynamic protein interactions in living plants.

• Plant synthetic biology: Employing antibodies to monitor the expression and activity of engineered proteins and metabolic pathways in plant systems.

• Environmental response monitoring: Developing antibody-based biosensors for real-time monitoring of plant protein responses to environmental stresses.

These emerging applications highlight the continuing importance of well-validated antibodies like the At3g55900 antibody in advancing plant biology research .

What resources and repositories can help researchers evaluate and select antibodies for plant research?

Researchers working with plant antibodies like the At3g55900 antibody can leverage several resources to improve experimental design and antibody selection:

• Antibody search engines and repositories:

  • CiteAb: Aggregates antibody citations to help identify validated antibodies

  • Antibodypedia: Collects validation data across applications

  • The Arabidopsis Information Resource (TAIR): Lists antibodies used in Arabidopsis research

• Validation resources:

  • Antibody Validation Database: Centralizes validation information

  • Plant Antibody Database: Specifically focused on plant research antibodies

  • Only Good Antibodies community: Discussion forum for antibody quality improvement

• Protocol repositories:

  • Bio-protocol: Detailed protocols for antibody-based applications in plant research

  • Protocol Exchange: Peer-reviewed protocols for plant protein detection methods

• Community standards:

  • International Working Group for Antibody Validation (IWGAV) guidelines

  • Adapted for plant research contexts

These resources help researchers make informed decisions about antibody selection and experimental design, contributing to more reproducible and reliable research outcomes .