At5g38270 Antibody

What is the At5g38270 protein and why is it studied in Arabidopsis thaliana?

At5g38270 is a protein encoded by the At5g38270 gene in Arabidopsis thaliana (Mouse-ear cress). While specific information about this protein's function requires further research, studying it contributes to our understanding of plant molecular biology. The corresponding antibody enables researchers to investigate protein expression patterns, subcellular localization, and potential interactions with other molecules .

What are the key specifications of commercially available At5g38270 Antibody?

At5g38270 Antibody is typically a polyclonal antibody raised in rabbits using recombinant Arabidopsis thaliana At5g38270 protein as the immunogen. It is antigen-affinity purified and provided in liquid form, stored in buffer containing glycerol and preservatives. The antibody is primarily validated for ELISA and Western Blot applications to ensure identification of the target antigen .

How does antibody validation ensure experimental reproducibility?

Antibody validation is crucial for reproducible research, especially given that approximately 50% of commercial antibodies fail to meet basic standards for characterization, resulting in billions in financial losses annually . Proper validation must demonstrate:

• The antibody binds specifically to At5g38270 protein

• It recognizes the target in complex mixtures (e.g., cell lysates)

• It does not cross-react with other proteins

• It performs consistently under specific experimental conditions

Validation should include knockout/knockdown controls, comparison with transcript data, and orthogonal detection methods.

What sample preparation protocol is optimal for At5g38270 detection in Arabidopsis tissues?

Based on protocols used for similar plant antibodies such as anti-ABI5 and anti-HY5, the following extraction method is recommended :

• Use fresh tissue or flash-freeze samples immediately after collection

• Extract with buffer containing: 50 mM Tris-HCl pH 8.0, 150-200 mM NaCl, 1% Triton X-100, 10 mM DTT, and protease inhibitor cocktail

• For seed samples (where expression may differ), modify the extraction buffer to 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 10 mM DTT, 1% Triton X-100, with protease inhibitor cocktail

• Denature samples in 4X SDS sample buffer at 95°C for 5 minutes

• Centrifuge at high speed (>10,000g) to remove cell debris before loading

What are the recommended Western blot conditions for At5g38270 Antibody?

For optimal Western blot results with At5g38270 Antibody, consider these evidence-based parameters from similar plant antibody research :

How should researchers design controls for experiments using At5g38270 Antibody?

Proper experimental controls are essential for antibody-based research and should include :

• Positive controls: Wild-type Arabidopsis thaliana tissues known to express At5g38270

• Negative controls:

  • Primary antibody omission

  • Knockdown/knockout mutant lines (if available)

  • Tissues where target protein is not expressed

• Treatment controls: For studies involving treatments affecting expression (e.g., hormones, stress), include appropriate treatment series

• Loading controls: Use established Arabidopsis loading controls such as anti-actin or anti-tubulin antibodies

What strategies optimize immunoprecipitation using At5g38270 Antibody?

Immunoprecipitation with At5g38270 Antibody requires careful optimization:

• Pre-clear lysates with Protein A/G beads to reduce non-specific binding

• Use 2-5 μg antibody per 500 μg of total protein

• Incubate overnight at 4°C with gentle rotation

• Optimize salt concentration in wash buffers (150-300 mM NaCl)

• Elute with low pH buffer or by boiling in SDS sample buffer

• Verify pull-down efficiency by Western blot analysis

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

How can researchers address weak or absent signals when using At5g38270 Antibody?

When experiencing detection issues with At5g38270 Antibody, consider these methodological adjustments:

How should researchers interpret multiple bands in Western blots with At5g38270 Antibody?

Multiple bands in Western blots require systematic analysis :

• Compare to predicted molecular weight: The expected size should be calculated based on amino acid sequence

• Consider post-translational modifications: Phosphorylation, glycosylation, or ubiquitination can alter mobility

• Evaluate potential degradation products: Partial degradation can produce specific fragments

• Assess splice variants: Alternative splicing can generate proteins of different sizes

• Test protein extraction conditions: Different buffers may preserve protein integrity differently

• Perform peptide competition assays: Specific bands should disappear when antibody is pre-incubated with immunizing peptide

How can researchers quantify Western blot results using At5g38270 Antibody for comparative analysis?

For reliable quantification in comparative studies:

How can At5g38270 Antibody be utilized in protein-protein interaction studies?

For investigating protein interactions involving At5g38270:

• Co-immunoprecipitation (Co-IP):

  • Optimize lysis conditions to maintain protein complexes

  • Use mild detergents (0.5-1% NP-40 or Triton X-100)

  • Include stabilizers like glycerol (10%) in buffers

  • Consider reversible crosslinking for transient interactions

• Proximity Ligation Assay (PLA):

  • Combines At5g38270 Antibody with antibodies against potential interaction partners

  • Requires high antibody specificity and optimization of fixation conditions

  • Provides spatial information about interaction locations

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Enables unbiased identification of interaction partners

  • Requires stringent controls (IgG, knockout samples)

  • Necessitates careful optimization of wash stringency

What considerations apply when using At5g38270 Antibody for immunohistochemistry/immunofluorescence?

While At5g38270 Antibody may not be explicitly validated for immunolocalization, researchers can optimize protocols:

• Test multiple fixation methods:

  • 4% paraformaldehyde for general protein preservation

  • Methanol/acetone for membrane proteins

  • Glutaraldehyde for challenging epitopes

• Optimize antigen retrieval:

  • Heat-induced epitope retrieval (citrate buffer, pH 6.0)

  • Enzymatic retrieval (proteinase K, trypsin)

  • Perform retrieval time-course to determine optimal conditions

• Antibody dilution testing:

  • Start with higher concentrations (1:50-1:200)

  • Include absorption controls with immunizing peptide

• Address plant-specific challenges:

  • Cell wall permeabilization optimization

  • Autofluorescence quenching (0.1% sodium borohydride or Sudan Black B)

  • Confocal settings to differentiate signal from background

How can computational approaches enhance At5g38270 antibody-based research?

In silico methods can significantly improve experimental design and interpretation:

• Epitope prediction and analysis:

  • Identify potential cross-reactivity with related proteins

  • Assess epitope conservation across species for evolutionary studies

  • Predict epitope accessibility in native protein

• Antibody-antigen complex modeling:

  • Simulate binding interface interactions

  • Predict effects of experimental conditions on binding

  • Optimize mutations to enhance binding affinity

• Expression analysis integration:

  • Correlate antibody detection with transcriptomic data

  • Identify tissues/conditions for optimal detection

  • Resolve discrepancies between protein and transcript levels

What minimum validation standards should be met before publishing results using At5g38270 Antibody?

Based on current reproducibility guidelines in antibody research :

• Demonstrate specificity:

  • Test with knockout/knockdown controls when available

  • Perform peptide competition assays

  • Validate across multiple experimental conditions

• Document experimental conditions:

  • Report complete antibody information (supplier, catalog number, lot, RRID)

  • Describe all experimental parameters (concentrations, incubation times, temperatures)

  • Provide detailed protocols as supplementary material

• Present appropriate controls:

  • Include loading controls for Western blots

  • Show full blots/gels, not just regions of interest

  • Document reproducibility across multiple experiments

• Validate key findings with orthogonal methods:

  • Confirm critical results with alternative approaches (e.g., GFP tagging, RNA analysis)

  • Test multiple antibodies targeting different epitopes when possible

How does batch-to-batch variation affect experimental outcomes with At5g38270 Antibody?

Polyclonal antibodies like At5g38270 Antibody can exhibit significant lot-to-lot variations that impact research:

• Sources of variation:

  • Different animal immune responses

  • Variations in antigen preparation

  • Changes in purification efficiency

• Mitigation strategies:

  • Record lot numbers and test new lots against previous ones

  • Maintain reference samples for comparative testing

  • Adjust protocols (dilution, incubation time) as needed for new lots

  • Consider creating large single-batch stocks for long-term projects

• Documentation requirements:

  • Report lot numbers in publications

  • Note any observed performance differences between batches

  • Document optimization adjustments for new lots

How can researchers address contradictory results between antibody detection and other methods?

When faced with discrepancies between At5g38270 Antibody results and other techniques:

• Assess technical factors:

  • Antibody specificity limitations

  • Sample preparation differences

  • Detection sensitivity thresholds

• Consider biological explanations:

  • Post-transcriptional regulation affecting transcript-protein correlation

  • Post-translational modifications altering epitope accessibility

  • Protein stability and turnover rates

  • Tissue-specific or developmental regulation

• Resolution approaches:

  • Employ multiple detection methods with different principles

  • Test different antibodies targeting different epitopes

  • Use genetic approaches (tagged proteins, knockouts)

  • Apply targeted proteomics approaches for validation