At4g33640 Antibody

What is At4g33640 and why is it significant for Arabidopsis thaliana research?

At4g33640 is a gene locus in Arabidopsis thaliana (mouse-ear cress) that encodes a protein identified by UniProt accession number Q8LBN7. The protein is studied in plant molecular biology research to understand plant cellular functions and regulatory pathways. Antibodies against this target are valuable tools for detecting and quantifying the protein's presence in various experimental conditions .

The significance of At4g33640 stems from its role in fundamental plant biological processes. When designing experiments with At4g33640 antibody, researchers should consider:

• The protein's normal expression levels in different tissues

• Its potential interactions with other proteins

• Subcellular localization patterns

• Changes in expression under different environmental conditions

A methodological approach to studying this protein begins with establishing baseline expression patterns using the antibody in wild-type plants before examining experimental conditions or genetic modifications.

What experimental applications are validated for At4g33640 Antibody?

At4g33640 Antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) applications . These techniques provide complementary approaches for protein detection:

For Western blot applications, researchers should optimize protocols by:

• Testing different protein extraction methods appropriate for plant tissues

• Determining optimal antibody dilutions (starting with manufacturer recommendations)

• Selecting appropriate blocking reagents to minimize background

• Including positive and negative controls to validate specificity

When using this antibody for identifying the At4g33640 protein in Arabidopsis extracts, proper sample preparation is critical for maintaining protein integrity and achieving reliable results.

How should At4g33640 Antibody be stored and handled to maintain optimal activity?

For maximum retention of immunoreactivity, At4g33640 Antibody should be stored at -20°C or -80°C immediately upon receipt . Researchers should follow these methodological guidelines:

• Aliquot the antibody into smaller volumes upon first thaw to prevent repeated freeze-thaw cycles

• Store working dilutions at 4°C for short-term use (within 1-2 weeks)

• When retrieving from freezer storage, thaw on ice rather than at room temperature

• Avoid vortexing the antibody solution; instead, mix gently by inversion or mild pipetting

• Centrifuge briefly before opening to collect solution at the bottom of the tube

The antibody is preserved in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . This formulation helps maintain stability during storage. When incorporating this antibody into experimental protocols, researchers should consider buffer compatibility with their assay systems.

What strategies can be employed to validate At4g33640 Antibody specificity in Arabidopsis thaliana research?

Antibody specificity is crucial for reliable experimental outcomes. For At4g33640 Antibody, comprehensive validation should include:

• Genetic Validation:

  • Testing the antibody in At4g33640 knockout/knockdown lines

  • Comparing signal in overexpression lines versus wild-type

  • Using CRISPR-edited plant lines with epitope modifications

• Biochemical Validation:

  • Performing peptide competition assays with the immunogen (recombinant At4g33640 protein)

  • Conducting immunoprecipitation followed by mass spectrometry

  • Testing cross-reactivity against related proteins in Arabidopsis

• Technical Controls:

  • Including secondary antibody-only controls

  • Testing pre-immune serum (if available) as a negative control

  • Comparing signals across multiple tissues with known expression patterns

While the antibody is produced using recombinant Arabidopsis thaliana At4g33640 protein as the immunogen , researchers should independently verify specificity, particularly when studying closely related protein families or when investigating previously unreported expression patterns.

How can researchers optimize immunohistochemistry protocols for At4g33640 detection in plant tissues?

While immunohistochemistry (IHC) is not listed among the validated applications for this antibody , researchers may adapt protocols for this purpose. A methodological approach includes:

• Tissue Preparation:

  • Fix tissues in 4% paraformaldehyde or another plant-appropriate fixative

  • Embed in paraffin or prepare for cryosectioning

  • Section tissues to 5-10 μm thickness for optimal antibody penetration

• Antigen Retrieval:

  • Test multiple antigen retrieval methods (heat-induced, enzymatic)

  • Optimize retrieval buffer pH and composition

  • Determine optimal retrieval duration

• Antibody Incubation:

  • Begin with 1:100 to 1:500 dilutions, then optimize

  • Incubate at 4°C overnight to improve signal-to-noise ratio

  • Test different blocking agents to reduce background

• Detection System:

  • Compare direct fluorescent conjugates versus amplification systems

  • Optimize counterstains for plant cell visualization

  • Include controls for autofluorescence, particularly important in plant tissues

For plant tissues specifically, researchers should consider cell wall permeabilization steps and account for natural autofluorescence when designing detection strategies.

What are the considerations for using At4g33640 Antibody in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) can reveal protein-protein interactions involving At4g33640. Key methodological considerations include:

• Lysis Buffer Optimization:

  • Test multiple lysis buffer compositions to preserve protein interactions

  • Consider mild detergents (0.1-0.5% NP-40 or Triton X-100)

  • Include protease and phosphatase inhibitors to maintain protein integrity

• Antibody Coupling:

  • Directly couple the antibody to protein A/G beads or magnetic beads

  • Determine optimal antibody-to-bead ratio

  • Consider crosslinking the antibody to beads to prevent co-elution

• Experimental Controls:

  • Include IgG control immunoprecipitations

  • Perform reverse Co-IPs when possible

  • Include input samples for comparison

• Elution and Analysis:

  • Optimize elution conditions to maintain complex integrity

  • Analyze by Western blot or mass spectrometry

  • Consider native elution for downstream functional assays

As this antibody is polyclonal , batch-to-batch variation may occur. Researchers should validate each new lot for Co-IP applications and consider using monoclonal antibodies for more standardized results if available.

How can researchers address weak or absent signals when using At4g33640 Antibody in Western blots?

When signal strength is suboptimal in Western blot applications, researchers should systematically evaluate:

• Protein Extraction Efficiency:

  • Test multiple extraction buffers optimized for plant tissues

  • Include adequate protease inhibitors to prevent degradation

  • Consider using specialized extraction kits for Arabidopsis

• Antibody Concentration and Incubation:

  • Increase antibody concentration (e.g., from 1:1000 to 1:500 or 1:250)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Test different diluents that may improve antibody performance

• Detection System Enhancement:

  • Use high-sensitivity detection substrates for HRP-conjugated secondary antibodies

  • Consider signal amplification systems

  • Optimize exposure times for digital imaging

• Sample Loading and Transfer:

  • Increase protein loading amount (up to 50-100 μg per lane)

  • Verify transfer efficiency using reversible staining

  • Adjust transfer conditions for high or low molecular weight proteins

As At4g33640 Antibody is antigen affinity-purified , it should provide specific detection when used at appropriate concentrations. Researchers should test different batches if persistent issues occur, as polyclonal antibodies may show batch-to-batch variation.

What strategies can researchers employ to minimize background in ELISA using At4g33640 Antibody?

Background reduction in ELISA requires methodical optimization:

• Blocking Optimization:

  • Test multiple blocking agents (BSA, casein, commercial blockers)

  • Extend blocking time (2-3 hours at room temperature)

  • Add 0.05% Tween-20 to washing and antibody diluent buffers

• Antibody Dilution Series:

  • Perform a checkerboard titration to determine optimal concentrations

  • Prepare antibody dilutions in blocking buffer

  • Include a pre-absorption step with irrelevant proteins

• Sample Preparation Refinement:

  • Further purify protein extracts before analysis

  • Pre-clear samples with protein A/G beads

  • Dialyze samples against ELISA buffer to remove interfering compounds

• Protocol Modifications:

  • Reduce incubation temperature (4°C instead of room temperature)

  • Include additional washing steps (5-7 washes instead of 3)

  • Consider plate types with different binding properties

A systematic approach to troubleshooting will help identify specific factors contributing to background and enable targeted optimization.

How can researchers adapt At4g33640 Antibody protocols for use in fluorescence microscopy?

Adapting At4g33640 Antibody for immunofluorescence requires special considerations for plant cells:

• Fixation Method Selection:

  • Compare aldehyde-based fixatives with organic solvent fixation

  • Optimize fixation duration to balance antigen preservation and accessibility

  • Include permeabilization steps appropriate for plant cell walls

• Antibody Delivery Optimization:

  • Test antibody penetration enhancers like Triton X-100 or saponin

  • Consider enzymatic cell wall digestion with cellulase/pectinase

  • Optimize incubation times for thick plant tissues

• Signal Detection Enhancement:

  • Use high-quantum-yield fluorophore-conjugated secondary antibodies

  • Implement signal amplification using tyramide or other systems

  • Employ spectral unmixing to distinguish signal from plant autofluorescence

• Mounting and Imaging:

  • Select anti-fade mounting media compatible with plant tissues

  • Use confocal microscopy to improve signal-to-noise ratio

  • Include appropriate negative and positive controls in the same imaging session

When adapting the antibody for immunofluorescence, researchers should be aware that this application is not listed among the manufacturer's validated uses and may require extensive optimization.

How should researchers design quantitative experiments using At4g33640 Antibody?

For quantitative analysis of At4g33640 protein levels, a rigorous experimental design should include:

• Standard Curve Generation:

  • Use purified recombinant At4g33640 protein at known concentrations

  • Generate a standard curve covering the expected concentration range

  • Ensure the curve encompasses the linear range of detection

• Sample Normalization:

  • Include housekeeping protein controls for Western blots

  • Normalize ELISA results to total protein concentration

  • Consider spike-in controls for recovery assessment

• Technical Replication:

  • Perform all measurements in triplicate at minimum

  • Include inter-assay controls across multiple experimental days

  • Calculate coefficients of variation to assess reproducibility

• Statistical Analysis:

  • Implement appropriate statistical tests based on experimental design

  • Account for multiple comparisons in complex experiments

  • Consider power analysis to determine required sample size

A sample data table for quantitative Western blot analysis might include:

What considerations should researchers address when planning long-term studies using At4g33640 Antibody?

For longitudinal studies spanning months or years, researchers should implement strategies to ensure consistent results:

• Antibody Lot Management:

  • Purchase sufficient antibody from a single lot for the entire study

  • Aliquot and store according to manufacturer recommendations

  • Validate each new lot against previous lots if replacements are needed

• Sample Collection Standardization:

  • Develop detailed SOPs for tissue collection and processing

  • Harvest samples at consistent times of day to control for circadian effects

  • Process all samples identically to minimize technical variation

• Data Normalization Across Timepoints:

  • Include internal reference samples that are analyzed at each timepoint

  • Maintain consistent positive and negative controls

  • Consider implementing a bridging study design for antibody lot changes

• Storage Considerations:

  • Determine optimal sample storage conditions for long-term stability

  • Evaluate the impact of freeze-thaw cycles on protein detection

  • Consider preparing master mixes of common reagents to minimize variation

Given the 14-16 week lead time for At4g33640 Antibody production , researchers should plan procurement well in advance of experimental needs and consider maintaining a reserve supply for critical experiments.

How can researchers integrate At4g33640 Antibody data with other molecular techniques for comprehensive analysis?

A multi-omics approach provides richer insights than antibody-based detection alone:

• Correlation with Transcriptomic Data:

  • Compare protein levels (antibody detection) with mRNA expression (RT-qPCR or RNA-seq)

  • Analyze potential post-transcriptional regulation mechanisms

  • Identify discrepancies that may indicate regulation at protein level

• Integration with Proteomics:

  • Validate antibody-based quantification with mass spectrometry data

  • Identify post-translational modifications not detected by the antibody

  • Map protein interaction networks through IP-MS approaches

• Functional Validation:

  • Complement protein detection with genetic manipulation (knockout/overexpression)

  • Correlate protein levels with phenotypic changes

  • Perform in vitro activity assays to link quantity to function

• Spatial Analysis Integration:

  • Combine immunolocalization with subcellular fractionation

  • Correlate tissue-specific expression with cell-type-specific transcriptomics

  • Analyze temporal-spatial regulation patterns

An integrated data analysis approach might be visualized as:

How can At4g33640 Antibody be adapted for high-throughput phenotypic screening in plant research?

Scaling antibody-based detection for high-throughput applications requires:

• Miniaturization Strategies:

  • Adapt protocols to 384- or 1536-well plate formats

  • Reduce required sample volumes through process optimization

  • Implement automated liquid handling for consistency

• Detection Method Modifications:

  • Develop homogeneous assay formats to reduce wash steps

  • Consider alternative detection technologies (e.g., AlphaLISA, HTRF)

  • Optimize signal development times for batch processing

• Quality Control Implementation:

  • Include standard controls on every plate

  • Calculate Z' factors to assess assay robustness

  • Implement statistical methods to identify positional plate effects

• Data Analysis Automation:

  • Develop automated image analysis pipelines for visual assays

  • Implement machine learning for complex phenotype recognition

  • Create standardized data processing workflows for consistency

While adapting At4g33640 Antibody for high-throughput applications, researchers should verify that assay performance remains comparable to standard formats, particularly regarding specificity and sensitivity.

What approaches can researchers use to study post-translational modifications of At4g33640 protein?

Detecting post-translational modifications (PTMs) requires specialized approaches:

• Modification-Specific Detection:

  • Combine At4g33640 Antibody with modification-specific antibodies

  • Use sequential immunoprecipitation to enrich modified forms

  • Employ multiplexed detection systems for simultaneous analysis

• Biochemical Enrichment:

  • Implement phosphopeptide enrichment techniques before analysis

  • Use lectin affinity for glycosylated forms

  • Apply SUMO/ubiquitin affinity purification for modified proteins

• Analytical Separation:

  • Utilize 2D gel electrophoresis to separate modified forms

  • Apply Phos-tag or similar technologies for phosphorylated protein separation

  • Implement isoelectric focusing to resolve charge variants

• Mass Spectrometry Integration:

  • Perform immunoprecipitation with At4g33640 Antibody followed by MS

  • Analyze PTM signatures through specialized MS fragmentation techniques

  • Quantify modification stoichiometry through targeted MS approaches

When studying PTMs, researchers should be aware that the specific epitope recognized by At4g33640 Antibody may be affected by modifications, potentially leading to altered detection sensitivity for modified forms.

How can researchers utilize At4g33640 Antibody in plant stress response studies?

For investigating At4g33640 protein's role in stress responses, researchers should consider:

• Stress Treatment Standardization:

  • Develop consistent stress application protocols

  • Include time-course analyses to capture dynamic responses

  • Compare multiple stress types to identify specific vs. general responses

• Subcellular Localization Changes:

  • Track protein redistribution following stress exposure

  • Implement fractionation protocols to quantify compartment-specific changes

  • Consider live cell imaging approaches for temporal resolution

• Protein Complex Dynamics:

  • Analyze stress-induced changes in protein interaction partners

  • Compare complex composition before and after stress application

  • Identify regulatory modifications triggered by stress conditions

• Correlation with Physiological Responses:

  • Link protein expression/modification patterns to physiological parameters

  • Integrate with metabolomic analyses for comprehensive stress response profiling

  • Compare wild-type and mutant responses to establish functional significance

This methodological approach enables researchers to establish whether At4g33640 is a stress response regulator, target, or component of adaptation mechanisms in Arabidopsis thaliana.

How might emerging antibody technologies enhance research applications of At4g33640 detection?

Advanced antibody technologies offer new research possibilities:

• Single-Domain Antibodies:

  • Smaller size enables better tissue penetration

  • Improved access to sterically hindered epitopes

  • Enhanced stability for challenging experimental conditions

• Proximity Labeling Applications:

  • Antibody-enzyme fusions for spatial proteomics

  • Identification of near-neighbor proteins in native context

  • Temporal control of labeling for dynamic interaction studies

• Intrabody Development:

  • Expression of antibody fragments within living plant cells

  • Real-time monitoring of protein dynamics

  • Targeted protein modulation through intrabody binding

• Multiplexed Detection Systems:

  • Simultaneous visualization of multiple proteins

  • Correlation of At4g33640 with interacting partners

  • Spatial relationship mapping at subcellular resolution

These emerging technologies may complement traditional applications of At4g33640 Antibody , enabling more detailed understanding of protein function and regulation in plant systems.

What computational approaches can enhance analysis of At4g33640 protein data from antibody-based experiments?

Computational methods improve data interpretation:

• Image Analysis Automation:

  • Machine learning for antibody staining pattern recognition

  • Automated quantification of signal intensity and distribution

  • Multi-parameter classification of cellular phenotypes

• Network Analysis Integration:

  • Incorporation of antibody-derived data into protein interaction networks

  • Pathway enrichment analysis to identify functional relationships

  • Causal network inference from perturbation experiments

• Structure-Function Prediction:

  • Epitope mapping through computational analysis

  • Structural modeling of protein regions detected by the antibody

  • Prediction of functional domains and their accessibility

• Multi-omics Data Integration:

  • Correlation analysis across different data types

  • Bayesian network modeling for causal relationship inference

  • Temporal trajectory analysis for dynamic processes

These computational approaches transform antibody-generated data from descriptive observations to mechanistic insights about At4g33640 protein function.

How can researchers contribute to improving research resources for At4g33640 protein studies?

The research community can enhance available resources through:

• Protocol Standardization and Sharing:

  • Publish detailed methodologies for successful applications

  • Deposit optimized protocols in repositories like protocols.io

  • Include comprehensive troubleshooting guides

• Validation Data Contribution:

  • Share antibody validation data in public repositories

  • Submit immunostaining images to appropriate databases

  • Report negative results to prevent duplication of effort

• Reagent Development:

  • Generate complementary tools (e.g., tagged constructs, reporter lines)

  • Develop application-specific antibody formats

  • Create knockout/knockdown resources for controls

• Data Integration:

  • Connect antibody-derived data with other Arabidopsis resources

  • Contribute to community databases for plant proteins

  • Link findings to gene ontology and functional annotations