At3g22845 Antibody

What is the At3g22845 protein in Arabidopsis thaliana?

At3g22845 refers to a specific protein encoded by the Arabidopsis thaliana genome, identified by its locus tag on chromosome 3. This protein (UniProt: Q9LIL4) is found in mouse-ear cress (Arabidopsis thaliana), a widely used model organism in plant molecular biology. While detailed functional characterization is still ongoing, antibodies against this protein enable researchers to study its expression patterns, interactions, and potential roles in plant cellular processes .

What are the key specifications of the At3g22845 Antibody?

The At3g22845 Antibody is a polyclonal antibody raised in rabbits using recombinant Arabidopsis thaliana At3g22845 protein as the immunogen. It is supplied in liquid form with 50% glycerol and 0.01M PBS (pH 7.4) buffer containing 0.03% Proclin 300 as a preservative. This antibody has been affinity-purified and is of IgG isotype. It is specifically designed to react with Arabidopsis thaliana specimens and has been validated for ELISA and Western Blot applications .

How should the At3g22845 Antibody be stored for optimal stability?

For optimal stability and performance, the At3g22845 Antibody should be stored at either -20°C or -80°C immediately upon receipt. The antibody is supplied in a storage buffer containing 50% glycerol, which helps prevent freezing damage. It's critical to avoid repeated freeze-thaw cycles as these can lead to protein denaturation and loss of antibody activity. When working with the antibody, aliquot the stock solution into smaller volumes to minimize freeze-thaw cycles for portions not immediately needed .

What are the validated experimental applications for At3g22845 Antibody?

The At3g22845 Antibody has been specifically validated for ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) applications. These techniques allow researchers to detect and quantify the target protein in complex samples. ELISA provides quantitative detection of the target in solution, while Western Blot enables visualization of the protein's molecular weight and relative abundance in tissue or cell lysates. Both applications ensure proper identification of the antigen when used with appropriate controls and optimization .

What is the recommended protocol for Western Blot using At3g22845 Antibody?

For optimal Western Blot results with At3g22845 Antibody, follow this methodological approach:

• Sample preparation:

  • Extract proteins from Arabidopsis tissues using an appropriate lysis buffer containing protease inhibitors

  • Quantify total protein concentration using Bradford or BCA assay

  • Prepare samples containing 20-50 μg total protein per lane

• Gel electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane (0.45 μm pore size recommended)

• Immunoblotting:

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

  • Dilute At3g22845 Antibody 1:1000 to 1:2000 in blocking solution

  • Incubate membrane with diluted antibody overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with HRP-conjugated anti-rabbit secondary antibody

  • Develop using chemiluminescent substrate and document results

• Controls:

  • Include wild-type and at3g22845 mutant/knockout samples when available

  • Consider using recombinant At3g22845 protein as a positive control

How can researchers optimize ELISA protocols using At3g22845 Antibody?

To optimize ELISA protocols with At3g22845 Antibody:

• Coating conditions:

  • Determine optimal antigen concentration (typically 1-10 μg/ml) for coating plates

  • Use carbonate/bicarbonate buffer (pH 9.6) for coating

  • Incubate overnight at 4°C for efficient binding

• Antibody dilution optimization:

  • Perform a titration series of At3g22845 Antibody (1:500 to 1:10,000)

  • Select the dilution providing the best signal-to-noise ratio

• Sample preparation:

  • Ensure consistent sample processing to minimize variability

  • Consider using calibration curves with recombinant protein standards

• Detection system:

  • Use HRP-conjugated anti-rabbit secondary antibody at manufacturer's recommended dilution

  • Develop with TMB substrate and measure absorbance at 450 nm

• Quality control:

  • Include negative controls (samples lacking target protein)

  • Run technical replicates to ensure reproducibility

How can researchers address specificity concerns when working with At3g22845 Antibody?

Addressing specificity concerns requires methodical validation:

• Experimental validation approaches:

  • Compare signal between wild-type and knockout/knockdown plant lines

  • Perform pre-adsorption tests using recombinant At3g22845 protein

  • Analyze peptide competition assays to confirm epitope specificity

• Cross-reactivity assessment:

  • Test the antibody against related Arabidopsis proteins

  • Examine signal in non-Arabidopsis plant species to confirm specificity

• Signal verification methods:

  • Compare detection patterns with published gene expression data

  • Validate results using orthogonal methods (e.g., mass spectrometry)

  • Confirm protein size matches predicted molecular weight

• Positive controls:

  • Use tissues known to express At3g22845 based on transcriptomic data

  • Consider testing overexpression lines as positive controls

What are common challenges in Western Blot experiments with At3g22845 Antibody and how can they be resolved?

Applying these troubleshooting approaches systematically will help resolve common technical issues when working with At3g22845 Antibody .

How should researchers interpret contradictory Western Blot results when using At3g22845 Antibody?

When faced with contradictory Western Blot results:

• Evaluate technical variables:

  • Verify consistent sample preparation methods across experiments

  • Confirm protein quantification accuracy using multiple methods

  • Assess gel percentage appropriateness for target protein size

  • Check transfer efficiency using reversible staining methods

• Biological considerations:

  • Examine if growth conditions, developmental stages, or treatments differed

  • Consider post-translational modifications affecting antibody recognition

  • Evaluate potential protein isoforms or splice variants

• Analytical approach:

  • Quantify band intensity using appropriate normalization controls

  • Perform statistical analysis across biological replicates

  • Compare results with published literature and database information

• Validation strategies:

  • Repeat experiments with additional controls

  • Implement orthogonal detection methods (immunoprecipitation, mass spectrometry)

  • Consider using alternative antibodies targeting different epitopes of At3g22845

How can At3g22845 Antibody be integrated with other techniques for comprehensive protein studies?

Integration of At3g22845 Antibody with complementary techniques enhances research depth:

• Immunohistochemistry/Immunofluorescence:

  • Optimize fixation conditions for plant tissues (4% paraformaldehyde recommended)

  • Test antibody dilutions between 1:100-1:500

  • Use confocal microscopy to determine subcellular localization of At3g22845

• Co-immunoprecipitation (Co-IP):

  • Use At3g22845 Antibody coupled to protein A/G beads

  • Identify interaction partners through mass spectrometry analysis

  • Verify interactions with reciprocal Co-IP experiments

• Chromatin Immunoprecipitation (ChIP):

  • If At3g22845 has DNA-binding properties, optimize crosslinking conditions

  • Enrich At3g22845-bound DNA fragments for sequencing analysis

  • Validate binding sites with reporter gene assays

• Proteomics integration:

  • Use immunoprecipitation followed by mass spectrometry

  • Compare protein abundance across different conditions

  • Identify post-translational modifications affecting function

What considerations should be made when designing experiments to study protein-protein interactions involving At3g22845?

When designing protein interaction studies:

• Experimental design considerations:

  • Select appropriate protein extraction buffers to preserve interactions

  • Consider native vs. denaturing conditions based on research questions

  • Include appropriate negative controls (IgG, unrelated antibodies)

• Co-IP optimization:

  • Test different lysis conditions (detergent types/concentrations)

  • Optimize antibody amounts and incubation times

  • Consider crosslinking to stabilize transient interactions

• Validation approaches:

  • Confirm interactions through reverse Co-IP experiments

  • Implement orthogonal methods (yeast two-hybrid, FRET)

  • Perform domain mapping to identify interaction interfaces

• Functional verification:

  • Test interaction relevance through mutational analysis

  • Assess interaction dynamics under different conditions

  • Investigate biological consequences of disrupting interactions

How can researchers apply At3g22845 Antibody in studies of plant stress responses or developmental processes?

Applying At3g22845 Antibody to study physiological processes:

• Experimental design for stress studies:

  • Expose plants to controlled stress conditions (drought, salt, pathogen)

  • Collect samples at multiple time points (0, 6, 12, 24, 48 hours)

  • Process samples consistently for protein extraction

  • Use At3g22845 Antibody to track protein abundance changes via Western Blot

• Developmental analysis approach:

  • Collect tissues from different developmental stages

  • Quantify At3g22845 protein levels relative to housekeeping controls

  • Correlate protein abundance with phenotypic changes

  • Compare with transcriptomic data to assess transcriptional vs. post-transcriptional regulation

• Tissue-specific expression:

  • Optimize immunohistochemistry protocols for different plant tissues

  • Map protein localization patterns throughout development

  • Compare with promoter-reporter studies for comprehensive expression analysis

• Functional studies:

  • Use antibody to verify knock-down efficiency in RNAi or CRISPR-edited plants

  • Assess protein abundance in overexpression lines

  • Correlate phenotypic effects with protein level alterations

What emerging technologies could enhance research applications of At3g22845 Antibody?

Emerging technologies offering new possibilities include:

• Advanced microscopy applications:

  • Super-resolution microscopy for precise subcellular localization

  • Live-cell imaging using nanobody derivatives

  • Correlative light and electron microscopy for ultrastructural context

• Proximity labeling approaches:

  • BioID or APEX2 fusions to identify proximal proteins in native cellular context

  • Spatially-resolved proteomic mapping within specific cellular compartments

  • Temporal tracking of protein interaction networks during stress response

• Single-cell applications:

  • Adapting antibody for single-cell western blot technologies

  • Integration with single-cell proteomics approaches

  • Spatial transcriptomics correlation with protein localization

• Computational integration:

  • Machine learning algorithms for automated Western Blot quantification

  • Systems biology approaches integrating antibody-derived data

  • Predictive modeling of protein function based on localization patterns

How might researchers validate custom modifications to standard protocols when working with At3g22845 Antibody?

When validating protocol modifications:

• Systematic optimization approach:

  • Modify only one variable at a time

  • Include standard protocol controls alongside modifications

  • Quantify signal-to-noise ratio improvements objectively

• Validation requirements:

  • Demonstrate reproducibility across multiple biological replicates

  • Verify specificity is maintained or improved

  • Ensure modifications do not introduce artifacts

• Documentation and reporting:

  • Record detailed procedural changes and supporting evidence

  • Include comparative data showing improvement over standard protocols

  • Specify critical parameters affecting reproducibility

• Cross-validation strategies:

  • Confirm findings using orthogonal detection methods

  • Verify results across different sample types

  • Test robustness across experimental conditions

What considerations should be made when comparing results from At3g22845 Antibody with gene expression data?

When integrating protein and transcriptomic data:

• Methodological considerations:

  • Account for temporal delays between transcription and translation

  • Consider post-transcriptional regulatory mechanisms

  • Normalize data appropriately for cross-platform comparisons

• Statistical approach:

  • Calculate correlation coefficients between mRNA and protein levels

  • Perform time-series analysis to identify regulatory patterns

  • Implement multivariate analysis to identify factors affecting correlation

• Biological interpretation:

  • Investigate discrepancies as potential regulatory mechanisms

  • Consider protein stability and turnover rates

  • Evaluate the impact of post-translational modifications

• Integrated analysis framework:

  • Develop computational workflows combining both data types

  • Visualize relationships through integrated heat maps or network diagrams

  • Identify condition-specific regulatory patterns through comparative analysis