At4g21240 Antibody

What is the At4g21240 gene and its encoded protein?

At4g21240 is a gene found in Arabidopsis thaliana (Mouse-ear cress), a widely used model organism in plant biology. The protein encoded by this gene is identified in the UniProt database with ID O49565. While the precise biological function of this protein remains under investigation, it represents an area of active research in plant biology. The protein's structural characteristics suggest potential roles in plant cellular processes, possibly including stress responses, development, or metabolic regulation. For researchers beginning work with this system, familiarization with the Arabidopsis Information Resource (TAIR) database is recommended for up-to-date annotations.

What are the key specifications of the At4g21240 Antibody?

The commercially available At4g21240 antibody has the following technical specifications:

This antibody is designed to bind specifically to the protein encoded by the At4g21240 gene. When planning experiments, researchers should consider these specifications alongside their specific research objectives and available equipment.

What are the primary applications of At4g21240 Antibody in plant research?

The At4g21240 antibody can be utilized for several key applications in plant research:

• Gene Expression Analysis: The antibody allows for localization of At4g21240 protein in different plant tissues, enabling spatial and temporal expression studies. This application typically involves immunohistochemistry or immunofluorescence techniques.

• Functional Studies: The antibody can be employed to investigate roles of the At4g21240 protein in various cellular processes, including potential involvement in stress responses, developmental pathways, or metabolic activities.

• Protein-Protein Interaction Mapping: Using co-immunoprecipitation techniques, researchers can identify protein binding partners of At4g21240, helping to elucidate its functional networks within plant cells.

These applications follow similar methodological principles to those established for other plant protein research, though specific protocol optimizations may be necessary.

How should At4g21240 Antibody be stored and handled?

Proper storage and handling of the At4g21240 antibody is critical for maintaining its efficacy:

• Storage Conditions: Lyophilized antibody preparations typically require -20°C storage in airtight containers to prevent moisture contamination. Once reconstituted, the antibody should be stored in small aliquots to minimize freeze-thaw cycles.

• Reconstitution Protocol: Follow manufacturer-specific guidelines for reconstitution. Generally, lyophilized antibodies should be reconstituted in sterile, nuclease-free water or appropriate buffer solutions.

• Working Dilutions: Establish optimal working dilutions for each application through preliminary experiments. Recommended starting dilutions for Western blot applications are approximately 1 μg/10 ml , though this may need adjustment for At4g21240 antibody specifically.

• Quality Control: Regularly assess antibody performance using positive controls to monitor potential degradation over time.

Proper documentation of storage conditions, reconstitution dates, and freeze-thaw cycles is advisable for troubleshooting variable results.

What controls should be used in experiments with At4g21240 Antibody?

Robust experimental design with appropriate controls is essential when working with At4g21240 antibody:

• Positive Controls: Include wild-type Arabidopsis samples known to express the At4g21240 protein.

• Negative Controls: Utilize At4g21240 knockout lines to validate target specificity. If these are unavailable, consider using tissues known not to express the protein.

• Secondary Antibody Controls: Include samples treated only with secondary antibody to identify non-specific binding.

• Blocking Peptide Controls: When available, use blocking peptides corresponding to the immunogen to confirm specificity.

• Loading Controls: For Western blots, include housekeeping proteins (e.g., actin or tubulin) to normalize expression levels across samples.

These controls should be processed identically to experimental samples to ensure valid comparisons and reliable results interpretation.

How can At4g21240 Antibody be used for protein localization studies?

For subcellular localization of the At4g21240 protein, researchers can implement the following methodological approach:

• Tissue Preparation: Fix plant tissues in 4% paraformaldehyde for 2-4 hours, followed by washing in PBS and either paraffin embedding for sectioning or clearing for whole-mount analysis.

• Antigen Retrieval: Apply heat-induced epitope retrieval using citrate buffer (pH 6.0) for optimal antibody binding, particularly with fixed tissues.

• Immunofluorescence Protocol:

  • Block sections with 5% normal serum in PBS with 0.1% Triton X-100 for 1 hour

  • Incubate with At4g21240 antibody at experimentally determined dilution (starting at 1:40)

  • Wash extensively with PBS-T (PBS with 0.1% Tween-20)

  • Apply fluorophore-conjugated secondary antibody

  • Counterstain nuclei with DAPI

  • Mount in anti-fade medium

• Confocal Microscopy: Capture images using appropriate excitation/emission wavelengths for the secondary antibody fluorophore. Z-stack imaging is recommended for comprehensive localization analysis.

• Colocalization Studies: Consider dual-labeling with established organelle markers to precisely define subcellular localization.

This methodology provides spatial information about protein distribution that complements expression data from biochemical approaches.

What are the best protocols for using At4g21240 Antibody in co-immunoprecipitation studies?

For investigating protein-protein interactions involving the At4g21240 protein:

• Sample Preparation:

  • Harvest 5-10g of Arabidopsis tissue and flash-freeze in liquid nitrogen

  • Grind to fine powder while maintaining frozen state

  • Add extraction buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitor cocktail)

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

  • Collect supernatant containing soluble proteins

• Pre-clearing:

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C with gentle rotation

  • Remove beads by centrifugation to reduce non-specific binding

• Immunoprecipitation:

  • Add At4g21240 antibody to pre-cleared lysate (2-5μg antibody per 1mg protein)

  • Incubate overnight at 4°C with gentle rotation

  • Add fresh Protein A/G beads and incubate for 2-4 hours

  • Wash beads 4-5 times with washing buffer (extraction buffer with reduced detergent)

  • Elute bound proteins with Laemmli buffer at 95°C for 5 minutes

• Analysis:

  • Separate proteins by SDS-PAGE

  • Confirm precipitation of At4g21240 protein by Western blot

  • Identify interacting partners by mass spectrometry or Western blotting with antibodies against suspected interactors

This protocol may require optimization based on protein abundance and solubility characteristics.

How can At4g21240 Antibody be validated for specificity in Arabidopsis thaliana?

Comprehensive validation of antibody specificity is critical for reliable experimental outcomes:

• Western Blot Validation:

  • Compare protein detection in wild-type versus At4g21240 knockout plants

  • Verify single band of expected molecular weight

  • Include blocking peptide competition to confirm specificity

  • Test cross-reactivity with closely related proteins if known

• Immunoprecipitation-Mass Spectrometry:

  • Perform immunoprecipitation as described above

  • Analyze precipitated proteins by mass spectrometry

  • Confirm enrichment of At4g21240 protein (UniProt ID: O49565)

  • Assess co-precipitation of known interactors versus non-specific binding

• Immunohistochemistry Controls:

  • Compare staining patterns between wild-type and knockout tissues

  • Perform peptide competition assays to confirm signal specificity

  • Include secondary antibody-only controls to assess background

• Recombinant Protein Testing:

  • Express recombinant At4g21240 protein in heterologous system

  • Confirm antibody recognition by Western blot

  • Test detection limits and linear range

These validation steps should be documented according to reporting guidelines for experimental protocols in life sciences .

What are the challenges in using At4g21240 Antibody for cross-species studies?

Cross-species applications present several methodological challenges:

• Sequence Homology Assessment:

  • Perform bioinformatic analysis to identify orthologs in target species

  • Align protein sequences to determine conservation of epitope regions

  • Predict potential cross-reactivity based on amino acid similarity

• Validation in Target Species:

  • Test antibody reactivity in tissues from target species

  • Compare band patterns with predicted molecular weights of orthologous proteins

  • Consider using overexpression or knockout systems if available in target species

• Optimization Strategies:

  • Adjust antibody concentration for each species

  • Modify buffer conditions to improve specificity

  • Test different blocking agents to reduce background

  • Consider alternative fixation methods for immunohistochemistry

• Interpretation Considerations:

  • Account for potential differences in protein expression levels

  • Consider post-translational modifications that may differ between species

  • Document all methodological adjustments and validation steps

While challenging, cross-species studies can provide valuable comparative insights when properly controlled and interpreted.

How can At4g21240 Antibody be used to investigate protein-protein interactions?

Beyond basic co-immunoprecipitation, several advanced approaches can be employed:

• Proximity Ligation Assay (PLA):

  • Fix and permeabilize plant tissues

  • Incubate with At4g21240 antibody and antibody against suspected interactor

  • Apply species-specific PLA probes

  • Perform ligation and rolling circle amplification

  • Visualize interaction signals by fluorescence microscopy

  • This technique allows visualization of interactions in situ with high sensitivity

• FRET-based Immunofluorescence:

  • Label At4g21240 antibody with donor fluorophore

  • Label interactor antibody with acceptor fluorophore

  • Analyze energy transfer using specialized microscopy

  • Calculate FRET efficiency to quantify interaction proximity

• Cross-linking Mass Spectrometry:

  • Apply membrane-permeable cross-linkers to intact plant tissues

  • Immunoprecipitate At4g21240 protein complexes

  • Analyze by mass spectrometry to identify cross-linked peptides

  • Map interaction interfaces with high resolution

• Co-fractionation Analysis:

  • Separate plant lysates by size exclusion chromatography

  • Analyze fractions by Western blot for At4g21240 and potential interactors

  • Identify co-elution patterns indicating complex formation

These approaches provide complementary information about the nature, context, and dynamics of protein interactions.

What methodologies can be used to interpret conflicting results with At4g21240 Antibody?

When facing experimental inconsistencies, systematic troubleshooting approaches should be implemented:

• Antibody Batch Variation Analysis:

  • Test multiple antibody lots if available

  • Quantify batch-to-batch variation in sensitivity and specificity

  • Document lot numbers associated with each experimental dataset

• Epitope Accessibility Assessment:

  • Compare different protein extraction methods (native vs. denaturing)

  • Test multiple fixation protocols for immunohistochemistry

  • Consider potential post-translational modifications that might mask epitopes

• Experimental Condition Optimization:

  • Systematically vary incubation times, temperatures, and buffer compositions

  • Document all parameters for reproducibility

  • Establish robust positive and negative controls for each condition

• Orthogonal Validation Approaches:

  • Corroborate antibody-based results with genetic approaches

  • Compare results with tagged protein expression systems

  • Correlate protein detection with mRNA expression data

• Statistical Analysis of Variability:

  • Implement appropriate statistical tests for replicate experiments

  • Consider power analysis to determine adequate sample sizes

  • Document natural biological variation versus technical variability

Transparent reporting of conflicting results and methodological approaches to resolve them advances the field more effectively than selective reporting of consistent findings alone.

How can At4g21240 Antibody be used in combination with other techniques for comprehensive protein analysis?

Integrative research approaches yield more robust insights:

• Multi-omics Integration:

  • Correlate protein localization/abundance with transcriptomic data

  • Compare interactome data with publicly available protein-protein interaction databases

  • Integrate with metabolomic data to connect protein function with metabolic pathways

• Genetic-Proteomic Correlation:

  • Analyze protein expression/localization in various mutant backgrounds

  • Compare phenotypic effects of gene knockout with protein distribution patterns

  • Use conditional expression systems to study temporal dynamics

• Structural Biology Integration:

  • Use antibody-based localization to guide sample preparation for structural studies

  • Correlate interaction data with available structural information

  • Consider epitope accessibility in the context of protein structure predictions

• High-throughput Applications:

  • Implement At4g21240 antibody in protein microarray studies

  • Develop tissue microarrays for systematic localization studies

  • Consider automated image analysis for quantitative immunohistochemistry

• In vivo Dynamics:

  • Complement antibody-based detection with live-cell imaging of fluorescently tagged proteins

  • Correlate fixed-sample immunolocalization with dynamic protein behavior

  • Use photoconvertible or photoactivatable tags to track protein movement

This integrative approach provides a more comprehensive understanding of protein function within complex biological systems.