At4g37235 Antibody

What is At4g37235 and what role does it play in plant biology?

At4g37235 (also known as AP22.102, CASP-like protein 5C1, or AtCASPL5C1) is a gene encoding a member of the Casparian strip membrane proteins (CASP) family in Arabidopsis thaliana. This protein is localized to the cell membrane as a multi-pass membrane protein, suggesting its involvement in membrane transport or signaling processes. The CASP family proteins are primarily known for their role in forming Casparian strips in plant roots, which are critical barrier-like structures that control the selective passage of water and nutrients.

What are the key characteristics of commercially available At4g37235 Antibody?

The At4g37235 Antibody is typically available as a liquid formulation preserved in 0.03% Proclin 300 with 50% Glycerol in 0.01M Phosphate Buffered Saline (PBS) at pH 7.4. Commercial preparations are often made-to-order with extended lead times (approximately 14-16 weeks), reflecting the specialized nature of this research reagent. The antibody targets the product of the At4g37235 gene, which has the UniProt accession number Q66GI1.

What experimental applications is At4g37235 Antibody suitable for?

The At4g37235 Antibody can be utilized in various molecular and cellular biology techniques, including:

• Western blotting for protein expression analysis

• Immunohistochemistry for localization studies in plant tissues

• Immunoprecipitation for protein-protein interaction studies

• Immunofluorescence for subcellular localization

• ELISA for quantitative protein detection

For optimal results in each application, researchers should conduct preliminary validation experiments to determine appropriate dilutions and conditions.

How should At4g37235 Antibody be stored and handled to maintain its activity?

To preserve antibody activity, At4g37235 Antibody should be stored according to manufacturer recommendations, typically at -20°C to -70°C for long-term storage . For working solutions, storage at 2-8°C under sterile conditions is generally appropriate for up to one month . Avoid repeated freeze-thaw cycles as these can significantly reduce antibody affinity and specificity. When handling the antibody, use sterile techniques and avoid contamination with microorganisms.

What controls should be included when using At4g37235 Antibody in Western blotting?

When designing Western blot experiments with At4g37235 Antibody, include the following controls:

• Positive control: Known sample expressing At4g37235 protein

• Negative control: Sample from knockout or knockdown plants

• Loading control: Detection of a constitutively expressed protein (e.g., actin or tubulin)

• Primary antibody control: Omitting primary antibody to detect non-specific binding

• Secondary antibody control: Using secondary antibody alone to detect background

• Blocking peptide control: Pre-incubating the antibody with the immunizing peptide

These controls help distinguish specific signals from background and validate antibody specificity.

How can immunohistochemistry protocols be optimized for At4g37235 detection in plant tissues?

For optimal immunohistochemistry results with At4g37235 Antibody:

• Fixation: Test different fixatives (4% paraformaldehyde, glutaraldehyde) to preserve epitope accessibility

• Antigen retrieval: Compare heat-induced and enzymatic methods

• Blocking: Use 3-5% BSA or normal serum from the species of the secondary antibody

• Antibody dilution: Test a range of antibody dilutions (typically starting at 1:100 to 1:1000)

• Incubation conditions: Compare overnight incubation at 4°C versus shorter periods at room temperature

• Detection system: Select appropriate secondary antibodies and visualization methods

• Counterstaining: Use suitable counterstains to provide tissue context

Document each parameter systematically to identify optimal conditions for specific plant tissues.

How does At4g37235 contribute to Casparian strip formation and function?

As a member of the CASP family, At4g37235 (AtCASPL5C1) likely contributes to the highly organized membrane domain formation necessary for Casparian strip development. The CASP proteins act as scaffolds that define the location of the Casparian strip by recruiting lignin biosynthesis machinery. Research methodologies to study this include:

• Confocal microscopy with fluorescently-tagged At4g37235 to track localization during Casparian strip formation

• Immunogold electron microscopy using At4g37235 Antibody to determine precise subcellular localization

• Co-immunoprecipitation with At4g37235 Antibody to identify interaction partners

• Genetic approaches using knockout/knockdown lines to assess functional significance

• Propidium iodide penetration assays to assess barrier function in mutant plants

These techniques help establish the spatial and temporal dynamics of At4g37235 during barrier formation.

What approaches can be used to study post-translational modifications of At4g37235?

To investigate post-translational modifications (PTMs) of At4g37235:

• Phosphorylation analysis:

  • Immunoprecipitate At4g37235 and analyze by phospho-specific antibodies

  • Use mass spectrometry to identify phosphorylation sites

  • Apply phosphatase inhibitors during protein extraction

• Glycosylation studies:

  • Use glycosidase treatments followed by Western blotting

  • Perform lectin binding assays

  • Employ mass spectrometry for glycan identification

• Ubiquitination detection:

  • Immunoprecipitate with At4g37235 Antibody and probe with anti-ubiquitin antibodies

  • Use proteasome inhibitors to accumulate ubiquitinated proteins

• Sequential extraction techniques to assess protein compartmentalization and modification states

These approaches can reveal regulatory mechanisms controlling At4g37235 function.

How can protein-protein interactions involving At4g37235 be characterized?

To identify and validate protein-protein interactions involving At4g37235:

• Co-immunoprecipitation (Co-IP):

  • Use At4g37235 Antibody to pull down protein complexes

  • Analyze interacting partners by mass spectrometry

• Proximity labeling techniques:

  • Express At4g37235 fused to BioID or APEX2

  • Identify nearby proteins through biotinylation and subsequent purification

• Yeast two-hybrid screening:

  • Use At4g37235 as bait to screen plant cDNA libraries

  • Validate interactions with directed Y2H assays

• Bimolecular Fluorescence Complementation (BiFC):

  • Express At4g37235 and candidate interactors as fusion proteins with split fluorescent protein fragments

  • Visualize interactions through reconstituted fluorescence

• Förster Resonance Energy Transfer (FRET):

  • Tag At4g37235 and potential partners with appropriate fluorophores

  • Measure energy transfer indicating molecular proximity

These methods provide complementary information about the At4g37235 interactome.

How can non-specific binding be reduced when using At4g37235 Antibody?

To minimize non-specific binding in immunoassays:

• Optimize blocking solutions:

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Adjust blocking time and temperature

• Improve antibody conditions:

  • Titrate antibody concentration to determine optimal dilution

  • Add 0.1-0.3% Triton X-100 or Tween-20 to reduce hydrophobic interactions

  • Pre-absorb antibody with plant extract from negative control samples

• Modify wash steps:

  • Increase number and duration of washes

  • Use higher salt concentration in wash buffers (150-500 mM NaCl)

• Filter primary antibody solution through 0.22 μm filter before use to remove aggregates

• For tissue sections, include an avidin/biotin blocking step if using biotin-based detection systems

Systematically test these modifications to determine which provides optimal signal-to-noise ratio.

What steps should be taken if At4g37235 Antibody shows declining performance over time?

If antibody performance deteriorates:

• Assess storage conditions:

  • Verify proper storage temperature

  • Check for evidence of contamination

  • Confirm absence of repeated freeze-thaw cycles

• Test activity with a reference sample:

  • Compare current results with historical positive controls

  • Quantify signal intensity decrease

• Perform antibody validation:

  • Re-test specificity using knockout/knockdown samples

  • Confirm epitope integrity using peptide competition

• Consider antibody stabilization:

  • Add carrier protein (BSA) to diluted antibody

  • Supplement with sodium azide (0.02%) as preservative

  • Aliquot stock solution to minimize freeze-thaw cycles

• If necessary, request a new lot of antibody and perform lot-to-lot comparison

Document all troubleshooting steps to identify the source of performance decline.

How can cross-reactivity of At4g37235 Antibody be assessed across different plant species?

To evaluate cross-reactivity across plant species:

• Sequence homology analysis:

  • Perform BLAST searches to identify At4g37235 homologs in target species

  • Align protein sequences to assess conservation of the antibody epitope

• Western blot validation:

  • Test protein extracts from multiple species in parallel

  • Include positive (Arabidopsis) and negative controls

  • Verify band size corresponds to predicted molecular weight in each species

• Immunoprecipitation followed by mass spectrometry:

  • Confirm pulled-down protein identity matches expected homolog

• Preabsorption control:

  • Preincubate antibody with recombinant proteins or synthetic peptides from target species

  • Compare immunostaining with and without preabsorption

• Validate with genetic approaches:

  • Test antibody on knockout/knockdown lines from related species if available

Cross-reactivity assessment is crucial for comparative studies across plant lineages.

How should variable expression patterns of At4g37235 across different plant tissues be interpreted?

When analyzing differential expression patterns:

• Establish baseline expression:

  • Quantify At4g37235 levels in multiple tissue types under standard conditions

  • Create expression maps showing relative protein abundance

• Consider developmental context:

  • Compare expression at different developmental stages

  • Correlate with known physiological transitions

• Analyze expression in response to environmental stimuli:

  • Test effects of abiotic stressors (drought, salinity, temperature)

  • Examine responses to biotic factors (pathogens, symbionts)

• Employ quantitative approaches:

  • Use densitometry for Western blots

  • Apply fluorescence intensity measurements for immunohistochemistry

  • Develop ELISA-based quantification methods

• Integrate with transcriptomic data:

  • Compare protein expression with mRNA levels

  • Identify potential post-transcriptional regulatory mechanisms

These analytical frameworks help distinguish biological variability from technical artifacts.

What statistical approaches are recommended for analyzing immunoblot data with At4g37235 Antibody?

For robust statistical analysis of immunoblot data:

• Experimental design considerations:

  • Use appropriate biological replicates (n≥3)

  • Include technical replicates for each biological sample

  • Randomize sample loading order

• Quantification methods:

  • Apply densitometry to measure band intensity

  • Use digital image analysis software with background subtraction

  • Normalize to loading controls (actin, tubulin, or total protein)

• Statistical tests:

  • For comparing two groups: t-test or Mann-Whitney U test

  • For multiple groups: ANOVA with appropriate post-hoc tests

  • For non-normally distributed data: non-parametric alternatives

• Data presentation:

  • Report fold-changes relative to control

  • Include error bars representing standard deviation or standard error

  • Present representative blots alongside quantification

• Reporting requirements:

  • Document antibody dilution, exposure times, and image processing steps

  • Specify normalization method and statistical tests applied

  • Report p-values and confidence intervals

These approaches enhance reproducibility and statistical validity of immunoblot analyses.

How can contradictory results between At4g37235 protein detection and gene expression data be reconciled?

When protein and transcript data appear inconsistent:

• Consider post-transcriptional regulation:

  • Assess mRNA stability using actinomycin D chase experiments

  • Investigate potential microRNA-mediated regulation

  • Examine alternative splicing patterns

• Evaluate post-translational mechanisms:

  • Measure protein half-life using cycloheximide chase assays

  • Assess proteasomal degradation with proteasome inhibitors

  • Investigate compartmentalization effects on protein detection

• Review methodological factors:

  • Check antibody specificity against recombinant protein standards

  • Verify primer specificity for transcript quantification

  • Compare extraction methods for protein and RNA

• Temporal considerations:

  • Perform time-course analyses to identify delays between transcription and translation

  • Sample at multiple time points following experimental treatments

• Integration approaches:

  • Use computational modeling to integrate transcriptomic and proteomic datasets

  • Apply systems biology frameworks to identify regulatory nodes

These strategies help identify biological mechanisms underlying transcript-protein discrepancies.

How can At4g37235 Antibody be utilized in high-throughput phenotypic screening approaches?

To implement At4g37235 Antibody in high-throughput screening:

• Automated immunoassay platforms:

  • Adapt to microplate-based ELISA formats

  • Develop high-content imaging workflows for immunofluorescence

  • Implement automated Western blot systems

• Multiplexing strategies:

  • Combine At4g37235 detection with other protein markers

  • Use differentially labeled secondary antibodies

  • Integrate with high-content imaging systems

• Flow cytometry applications:

  • Detect At4g37235 in protoplast preparations

  • Combine with other cellular markers

  • Sort cells based on expression levels

• Screening platform adaptation:

  • Optimize for 96 or 384-well formats

  • Develop standardized protocols for consistent detection

  • Implement automated image analysis algorithms

• Data management:

  • Create analysis pipelines for large-scale data processing

  • Implement machine learning for pattern recognition

  • Develop databases for phenotypic correlations

These approaches enable systematic exploration of At4g37235 function across genetic or environmental variables.

What role might computational modeling play in understanding At4g37235 function based on antibody-derived data?

Computational approaches to leverage antibody-derived data include:

• Structural modeling:

  • Predict 3D structure of At4g37235 using homology modeling

  • Simulate membrane integration based on localization data

  • Model structural changes under different conditions

• Network analysis:

  • Construct protein-protein interaction networks centered on At4g37235

  • Identify functional modules and pathways

  • Predict cellular outcomes of perturbations

• Integration with multi-omics data:

  • Correlate protein expression with transcriptomic, metabolomic data

  • Develop mathematical models of regulatory circuits

  • Simulate cellular responses to environmental stimuli

• Machine learning applications:

  • Classify cellular phenotypes based on immunostaining patterns

  • Predict protein function from localization data

  • Identify novel regulatory relationships

• Virtual screening:

  • Model binding sites for potential interacting partners

  • Simulate effects of post-translational modifications

  • Predict functional consequences of genetic variants

These computational approaches complement experimental data to generate testable hypotheses about At4g37235 function.

What validation standards should be applied when using At4g37235 Antibody in published research?

For rigorous validation in published studies:

• Specificity controls:

  • Test on knockout/knockdown lines

  • Peptide competition assays

  • Detect recombinant protein at appropriate molecular weight

• Application-specific validation:

  • For Western blotting: include molecular weight markers and loading controls

  • For immunohistochemistry: show negative controls and known expression patterns

  • For immunoprecipitation: demonstrate enrichment compared to input

• Reproducibility standards:

  • Use multiple biological replicates (minimum n=3)

  • Test multiple antibody lots when possible

  • Validate key findings with complementary techniques

• Reporting requirements:

  • Document complete antibody information (catalog number, lot, dilution)

  • Specify exact experimental conditions

  • Include all control experiments in supplementary materials

• Method transparency:

  • Provide detailed protocols for antibody use

  • Describe image acquisition and processing methods

  • Make original, unprocessed images available

These standards enhance research reproducibility and facilitate comparison across studies.

How can researchers ensure consistent results across different lots of At4g37235 Antibody?

To maintain consistency across antibody lots:

• Lot comparison strategy:

  • Test new lots side-by-side with previous lot

  • Generate standard curves for quantitative applications

  • Document optimal working dilutions for each lot

• Reference sample system:

  • Maintain frozen aliquots of positive control samples

  • Create standardized lysates as reference standards

  • Develop recombinant protein standards for calibration

• Validation protocol:

  • Test each new lot on known positive and negative samples

  • Verify expected staining pattern in key tissues

  • Confirm specificity through peptide competition

• Documentation practices:

  • Record lot-specific performance characteristics

  • Maintain detailed protocol adjustments for each lot

  • Document images from standard samples for comparison

• Supplier communication:

  • Request certificate of analysis for each lot

  • Inquire about manufacturing changes

  • Report inconsistencies to manufacturer