At3g06390 Antibody

What protein does the At3g06390 antibody target in Arabidopsis?

At3g06390 antibodies target proteins encoded by the At3g06390 gene locus in Arabidopsis thaliana. This antibody recognizes specific epitope structures within plant cell wall components, similar to how the MAC207 antibody recognizes arabinogalactan proteins in various plant species. The antibody-antigen interaction is highly specific, making it valuable for investigating protein expression and localization in plant tissues. When designing experiments with this antibody, researchers should consider both the protein's predicted molecular weight and potential post-translational modifications that may affect recognition.

What are the primary research applications for At3g06390 antibody?

The At3g06390 antibody serves multiple research purposes similar to other plant protein-specific antibodies:

• Immunolocalization studies to determine spatial distribution of the target protein in plant tissues

• Western blot analysis for protein expression quantification and molecular weight confirmation

• Immunoprecipitation for protein complex isolation and interaction studies

• Flow cytometry for cell population analysis expressing the target protein

• Chromatin immunoprecipitation (ChIP) if the target has DNA-binding properties

For optimal results, researchers should verify the antibody's performance in their specific experimental conditions, as the working concentration may vary depending on the application and tissue type .

How should At3g06390 antibody samples be stored to maintain activity?

For optimal longevity and performance, At3g06390 antibodies should be stored following these guidelines:

• Store antibody aliquots at -20°C for long-term storage (similar to other research antibodies like MAC207)

• Avoid repeated freeze-thaw cycles by preparing small working aliquots

• For short-term use (1-2 weeks), storage at 4°C with appropriate preservatives is acceptable

• Protect from direct light exposure, especially if conjugated to fluorophores

• If supplied as hybridoma supernatant, follow specific storage conditions provided by the manufacturer

Regular validation of antibody activity after extended storage periods is recommended using positive control samples to ensure consistent performance across experiments .

How can I optimize immunostaining protocols for At3g06390 antibody in different plant tissues?

Optimizing immunostaining with At3g06390 antibody requires systematic adjustment of multiple parameters:

• Fixation method: For plant tissues, a comparison between 4% paraformaldehyde, glutaraldehyde, and ethanol-based fixatives should be performed to determine optimal epitope preservation while maintaining tissue morphology.

• Antigen retrieval: Test both heat-mediated and enzymatic antigen retrieval methods if initial staining yields weak signals. For plant cell wall proteins, enzymatic digestion with pectinase or cellulase may improve antibody accessibility.

• Blocking solution optimization: Compare different blocking agents (BSA, normal serum, milk proteins) at concentrations ranging from 1-5% to reduce background.

• Antibody dilution series: Establish an optimal dilution range through a titration experiment (typically 1:100 to 1:2000) for both primary and secondary antibodies.

• Incubation conditions: Compare results between overnight incubation at 4°C versus shorter incubations (2-4 hours) at room temperature.

Document all optimization steps systematically and include appropriate controls (no primary antibody, pre-immune serum, competitive inhibition with antigen) .

What approaches can resolve contradictory data when At3g06390 antibody shows unexpected patterns in mutant lines?

When facing contradictory results with At3g06390 antibody in mutant lines, implement this systematic troubleshooting approach:

• Verify antibody specificity through alternative methods:

  • Perform western blots comparing wild-type and knockout mutants

  • Test competitive inhibition with purified antigen

  • Validate with a second antibody targeting a different epitope on the same protein

• Genetic verification:

  • Confirm genotype of mutant lines through PCR-based genotyping

  • Verify transcript levels using qRT-PCR

  • Consider alternative splicing or incomplete knockout possibilities

• Cross-reactivity assessment:

  • Test for recognition of homologous proteins in the same family

  • Perform epitope mapping to identify potential cross-reactive domains

• Technical validation:

  • Employ multiple detection methods (fluorescence, chromogenic, etc.)

  • Include positive control samples from verified sources

  • Compare results across different tissue fixation and sample preparation methods

Document all validation steps and consider alternative interpretations of the data, including potential compensatory mechanisms in mutant lines or unexpected protein interactions .

How can I quantitatively assess At3g06390 protein expression across developmental stages?

For quantitative assessment of At3g06390 protein expression across developmental stages, implement this comprehensive methodology:

• Sampling strategy:

  • Collect tissues at defined developmental time points using standardized growth conditions

  • Ensure biological replicates (minimum n=3) for statistical validity

  • Include both spatial (different tissues) and temporal (different stages) sampling

• Protein extraction optimization:

  • Compare extraction buffers containing different detergents (Triton X-100, NP-40, SDS)

  • Evaluate the necessity of protease inhibitors and phosphatase inhibitors

  • Optimize tissue disruption methods (grinding, sonication, pressure homogenization)

• Quantification methods:

  • Western blot analysis with appropriate loading controls (housekeeping proteins)

  • ELISA for absolute quantification

  • Immunohistochemistry with digital image analysis for spatial distribution

• Data analysis:

  • Normalize expression data to total protein or specific reference proteins

  • Apply appropriate statistical tests for comparing developmental stages

  • Generate expression profiles with error bars representing biological variation

Note: This table provides a hypothetical expression pattern framework for experimental design. Actual values should be determined experimentally .

What is the epitope specificity and cross-reactivity profile of At3g06390 antibody?

Understanding the epitope specificity and cross-reactivity profile is crucial for interpreting experimental results with At3g06390 antibody:

The epitope recognition pattern would likely follow similar principles as observed with plant cell wall antibodies like MAC207, which recognizes specific carbohydrate structures such as (β)GlcA1→3(α)GalA1→2Rha motifs in arabinogalactan proteins . For At3g06390 antibody, specificity testing should include:

• Epitope mapping through:

  • Peptide arrays covering the full protein sequence

  • Deletion mutant analysis

  • Competitive binding assays with synthesized peptides

• Cross-reactivity assessment:

  • Testing against recombinant proteins from related gene family members

  • Evaluation in multiple plant species to determine conservation

  • Analysis in various tissue types to identify potential non-specific binding

• Performance in different applications:

  • Western blot detection limit and specificity

  • Immunoprecipitation efficiency

  • Immunohistochemistry background levels

This information helps researchers interpret signals in complex samples and design appropriate controls for experiments .

How does phosphorylation status affect At3g06390 antibody recognition?

The effect of phosphorylation on antibody recognition is a critical consideration for researchers working with At3g06390 antibody:

• Epitope masking effects:

  • If the antibody epitope contains potential phosphorylation sites, phosphorylation may directly block antibody binding

  • Conformational changes induced by phosphorylation at distant sites may indirectly affect epitope accessibility

• Experimental approaches to assess phosphorylation effects:

  • Compare antibody binding to samples treated with/without phosphatase inhibitors

  • Perform parallel detection with phospho-specific and total protein antibodies

  • Use lambda phosphatase treatment to systematically dephosphorylate samples

• Analytical strategies:

  • 2D gel electrophoresis to separate phosphorylated isoforms before western blotting

  • Phospho-enrichment methods (IMAC, TiO₂) followed by immunoprecipitation

  • Mass spectrometry validation of phosphorylation status in immunoprecipitated samples

• Interpretation considerations:

  • Signal intensity changes may reflect phosphorylation status rather than total protein abundance

  • Different results across sample types may indicate tissue-specific phosphorylation patterns

  • Temporal changes may correlate with activation of specific kinase pathways

This information helps researchers distinguish between actual protein level changes and modifications affecting antibody recognition .

How can I resolve weak or inconsistent signals when using At3g06390 antibody in western blots?

Addressing weak or inconsistent western blot signals with At3g06390 antibody requires a systematic troubleshooting approach:

• Sample preparation optimization:

  • Test different extraction buffers (varying detergents, salt concentrations)

  • Evaluate different sample heating conditions (60°C, 70°C, 95°C)

  • Compare reducing vs. non-reducing conditions if disulfide bonds affect epitope structure

  • Include protease inhibitors to prevent degradation during extraction

• Transfer efficiency improvement:

  • Optimize transfer conditions for high molecular weight proteins (if applicable)

  • Test different membrane types (PVDF vs. nitrocellulose)

  • Evaluate wet transfer vs. semi-dry transfer methods

  • Consider using transfer buffer with reduced methanol for larger proteins

• Detection sensitivity enhancement:

  • Increase antibody concentration incrementally (1:2000, 1:1000, 1:500)

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

  • Test more sensitive detection methods (chemiluminescence vs. fluorescence)

  • Use signal enhancement systems (biotin-streptavidin amplification)

• Background reduction strategies:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Increase washing duration and detergent concentration

  • Pre-absorb antibody with plant extract from knockout tissue

  • Use more stringent blocking conditions (longer time, higher concentration)

This systematic approach should help identify the specific factors affecting antibody performance in your experimental system .

What controls are essential when using At3g06390 antibody for co-localization studies?

Rigorous controls are essential for reliable co-localization studies using At3g06390 antibody:

• Primary antibody controls:

  • Omission of primary antibody to assess secondary antibody specificity

  • Pre-immune serum control at equivalent concentration

  • Antibody pre-absorption with purified antigen

  • Use of knockout/knockdown tissues as negative controls

• Fluorophore and channel cross-talk controls:

  • Single fluorophore controls to establish bleed-through parameters

  • Secondary antibody cross-reactivity assessment

  • Autofluorescence controls (untreated samples)

  • Fluorescence quenching controls for sequential imaging

• Co-localization specific controls:

  • Known non-colocalizing proteins (negative control)

  • Known colocalizing proteins (positive control)

  • Randomized image analysis to establish background colocalization coefficients

  • Analysis of regions lacking the structure of interest

• Quantitative validation:

  • Multiple statistical measures (Pearson's, Manders' coefficients)

  • Analysis across multiple cells and experiments

  • Colocalization in multiple z-planes for 3D confirmation

  • Line scan analysis across subcellular structures

These controls ensure that observed colocalization patterns represent genuine biological associations rather than technical artifacts .

How should I design experiments to validate At3g06390 antibody specificity in new experimental systems?

To validate At3g06390 antibody specificity in new experimental systems, implement this comprehensive approach:

• Genetic validation strategy:

  • Compare antibody reactivity in wild-type vs. knockout/knockdown lines

  • Test overexpression lines for increased signal intensity

  • Use CRISPR-edited lines with epitope modifications

  • Compare multiple independent mutant alleles affecting the same gene

• Biochemical validation methods:

  • Immunoprecipitation followed by mass spectrometry identification

  • Western blot analysis of recombinant protein and native samples

  • Competitive inhibition with purified antigen at increasing concentrations

  • Size comparison with predicted molecular weight accounting for modifications

• Cellular/tissue validation approaches:

  • Compare immunostaining patterns with fluorescent protein fusions

  • Evaluate consistency of localization patterns with known biology

  • Test antibody in heterologous expression systems

  • Compare staining patterns with in situ hybridization of mRNA

• Cross-species validation:

  • Test reactivity in closely related species with conserved proteins

  • Evaluate reactivity in divergent species with low sequence homology

  • Correlate signal intensity with evolutionary distance

  • Compare sequence conservation at epitope regions

• Documentation standards:

  • Maintain detailed records of all validation experiments

  • Document antibody lot numbers used for validation

  • Record specific conditions that affect antibody performance

  • Share validation data when publishing results

This comprehensive validation approach ensures that experimental findings can be confidently attributed to the protein of interest rather than artifacts or cross-reactivity .

What is the optimal protocol for using At3g06390 antibody in chromatin immunoprecipitation (ChIP) experiments?

For optimal ChIP experiments using At3g06390 antibody, follow this methodological framework:

• Chromatin preparation:

  • Cross-link plant tissue with 1% formaldehyde for 10-15 minutes at room temperature

  • Quench with 0.125M glycine for 5 minutes

  • Isolate nuclei using sucrose gradient centrifugation

  • Sonicate chromatin to fragments of 200-500bp (optimize cycles empirically)

  • Verify fragment size by agarose gel electrophoresis

• Immunoprecipitation optimization:

  • Pre-clear chromatin with protein A/G beads

  • Test antibody amounts (2-10μg per reaction)

  • Compare different incubation conditions (4°C overnight vs. room temperature for 4 hours)

  • Include appropriate controls (IgG control, no antibody control, input sample)

• Washing and elution:

  • Implement stringent washing with increasing salt concentrations

  • Optimize wash buffer compositions based on signal-to-noise ratio

  • Elute protein-DNA complexes with SDS buffer at 65°C

  • Reverse cross-links overnight at 65°C with proteinase K treatment

• Analysis methods:

  • Perform qPCR on known target regions and negative control regions

  • Calculate enrichment as percent of input and relative to IgG control

  • Consider sequencing (ChIP-seq) for genome-wide binding analysis

  • Validate findings with biological replicates and alternative antibodies if available

This protocol should be optimized for each specific experimental system, with particular attention to sonication conditions and antibody concentrations .

How can I effectively use At3g06390 antibody for immunoprecipitation followed by mass spectrometry (IP-MS)?

For effective immunoprecipitation-mass spectrometry (IP-MS) using At3g06390 antibody, implement this optimized protocol:

• Sample preparation considerations:

  • Harvest tissue quickly and flash-freeze in liquid nitrogen

  • Optimize extraction buffer (typically containing 0.1-1% NP-40 or Triton X-100)

  • Include protease inhibitors, phosphatase inhibitors, and EDTA

  • Perform extraction at 4°C to minimize protein degradation

• Immunoprecipitation strategy:

  • Compare direct antibody coupling to beads vs. indirect capture

  • Test different antibody-to-sample ratios (typically 1-10μg antibody per mg protein)

  • Optimize incubation time (2-16 hours) and temperature (4°C)

  • Include stringent controls (IgG control, knockout tissue, competing peptide)

• Washing optimization:

  • Develop a washing strategy that balances background reduction with complex preservation

  • Test detergent concentration reduction in sequential washes

  • Consider cross-linking antibody to beads to prevent antibody contamination

  • Perform final washes in MS-compatible buffers without detergents

• MS-specific considerations:

  • Elute in conditions compatible with downstream MS analysis

  • Digest samples with high-quality trypsin or alternative proteases

  • Include replicate samples for statistical validation

  • Consider SILAC or TMT labeling for quantitative comparison

• Data analysis approach:

  • Filter against appropriate negative controls

  • Apply stringent statistical criteria (typically fold change >2, p<0.05)

  • Validate key interactions through orthogonal methods (yeast two-hybrid, BiFC)

  • Perform functional enrichment analysis on identified interactors

This comprehensive approach maximizes the identification of genuine protein interactions while minimizing experimental artifacts .