At2g33705 Antibody

What is the At2g33705 protein in Arabidopsis thaliana?

At2g33705 (UniProt accession Q3EBP0) is a protein found in Arabidopsis thaliana (Mouse-ear cress). This protein is still being characterized in current research, with antibodies developed to study its expression patterns and functions in plant cellular processes. When working with the antibody, researchers should be aware that proper validation is essential as antibody specificity issues have been documented for other targets .

What are the validated applications for At2g33705 Antibody?

At2g33705 Antibody has been validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications. These techniques enable researchers to detect and quantify the presence of the target protein in plant tissue extracts. The antibody has been affinity-purified to ensure specific binding to the recombinant Arabidopsis thaliana At2g33705 protein used as the immunogen .

What are the optimal storage conditions for At2g33705 Antibody?

For optimal preservation of antibody function, store At2g33705 Antibody at -20°C or -80°C upon receipt. The antibody is supplied in liquid form containing 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative. Avoid repeated freeze-thaw cycles as these can compromise antibody functionality and specificity .

How should I validate At2g33705 Antibody specificity for my experiments?

Antibody validation is crucial given the documented problems with antibody specificity in the literature . Recommended validation steps include:

• Positive and negative controls: Include wild-type Arabidopsis tissue (positive control) and knockout/knockdown lines lacking At2g33705 expression (negative control)

• Multiple detection methods: Validate findings using both Western blot and ELISA

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide to confirm binding specificity

• Cross-reactivity testing: Test the antibody against closely related proteins to assess potential cross-reactivity

These validation steps are essential as research has shown that commercial antibodies can sometimes bind to proteins other than their intended targets .

What experimental controls should be included when working with At2g33705 Antibody?

A robust experimental design should include the following controls:

Implementing these controls addresses the specificity concerns documented with other antibodies, where several antibodies raised against the same protein produced different banding patterns in Western blots .

What is the optimal protocol for Western blot using At2g33705 Antibody?

Based on antibody specifications and general best practices for plant protein analysis:

• Sample preparation:

  • Homogenize plant tissue in extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitors)

  • Centrifuge at 12,000g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• Western blot procedure:

  • Separate 20-50 μg of protein by SDS-PAGE (10-12% gel)

  • Transfer to PVDF membrane (100V for 1 hour)

  • Block with 5% non-fat milk in TBST for 1 hour

  • Incubate with At2g33705 Antibody (1:1000 dilution) overnight at 4°C

  • Wash 3× with TBST (10 minutes each)

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour

  • Wash 3× with TBST

  • Develop using ECL substrate

• Image analysis:

  • Document results using imaging system

  • Quantify band intensity using appropriate software

This protocol incorporates quality control measures to address issues of non-specific binding documented with other antibodies .

How should the At2g33705 Antibody be optimized for immunohistochemistry applications?

While the antibody specifications primarily list ELISA and Western blot applications , researchers may wish to adapt it for immunohistochemistry:

• Tissue fixation and embedding:

  • Fix tissue in 4% paraformaldehyde for 24 hours

  • Dehydrate through ethanol series

  • Embed in paraffin or freeze in OCT compound

• Antibody optimization steps:

  • Test multiple antibody concentrations (1:100, 1:250, 1:500, 1:1000)

  • Compare antigen retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Optimize incubation times (1-hour room temperature vs. overnight at 4°C)

  • Test detection systems (fluorescent vs. HRP-based)

• Validation controls:

  • Include peptide competition control

  • Test on confirmed At2g33705 knockout tissue

This methodical approach is essential since antibody cross-reactivity can lead to misidentification of target proteins, as documented in other research systems .

How can I distinguish between specific and non-specific signals when using At2g33705 Antibody?

Distinguishing specific from non-specific signals requires systematic analysis:

• Expected molecular weight: The predicted molecular weight of At2g33705 should be confirmed through bioinformatic analysis. Post-translational modifications may alter the observed molecular weight.

• Pattern analysis: Compare observed banding patterns with published results. Multiple bands could indicate:

  • Protein isoforms

  • Post-translational modifications

  • Degradation products

  • Non-specific binding

• Decisive validation tests:

  • Genetic knockout controls (signal should disappear)

  • Peptide competition assay (signal should be blocked)

  • Signal comparison across different tissues (should correlate with known expression patterns)

This approach addresses the challenges observed in research where different antibodies targeting the same protein produced vastly different banding patterns .

What are the common causes of false positives/negatives with At2g33705 Antibody and how can they be addressed?

This troubleshooting guide addresses issues similar to those documented in studies showing that antibodies can produce inconsistent results even when targeting the same epitope .

How can At2g33705 Antibody be utilized in protein-protein interaction studies?

Advanced protein-protein interaction studies can be conducted using the following approaches:

• Co-immunoprecipitation (Co-IP):

  • Lyse plant tissues in non-denaturing buffer

  • Pre-clear lysate with Protein A/G beads

  • Incubate with At2g33705 Antibody (5 μg per 1 mg protein)

  • Capture complexes with Protein A/G beads

  • Wash extensively

  • Elute and analyze by mass spectrometry or Western blot

• Proximity ligation assay (PLA):

  • Fix plant tissues or cells

  • Incubate with At2g33705 Antibody and antibody against potential interactor

  • Apply PLA probes and ligase

  • Amplify signal and visualize interaction sites

• FRET-based approaches:

  • Label At2g33705 Antibody with donor fluorophore

  • Label second antibody with acceptor fluorophore

  • Analyze energy transfer indicating proximity

These methodologies should be validated with appropriate controls to ensure specificity, which is a critical concern based on antibody specificity studies .

What computational approaches can complement At2g33705 Antibody research?

Integrating computational methods with antibody-based experiments can enhance research outcomes:

• Epitope prediction and analysis:

  • Analyze the immunogen sequence used to generate the antibody

  • Predict potential cross-reactive epitopes in the proteome

  • Map epitopes to protein structures when available

• Deep learning for antibody imaging analysis:

  • Apply machine learning algorithms to quantify immunohistochemistry results

  • Identify subtle expression patterns not evident through visual inspection

  • Compare with deep learning approaches being developed for antibody design

• Structural modeling of antibody-antigen interactions:

  • Generate computational models of At2g33705 structure

  • Predict antibody binding sites

  • Compare with crystal structures of similar antibody-antigen complexes

This integration of computational and experimental approaches addresses challenges similar to those being tackled in de novo antibody design research .

How can I adapt At2g33705 Antibody for high-throughput screening applications?

For large-scale studies across multiple plant samples or conditions:

• Antibody microarray development:

  • Spot At2g33705 Antibody on activated glass slides

  • Incubate with fluorescently labeled protein extracts

  • Scan and quantify signal intensities

• Automated Western blot analysis:

  • Utilize capillary-based automated Western systems

  • Optimize antibody concentration for the system

  • Develop standardized analysis protocols

• High-content imaging:

  • Combine At2g33705 Antibody with other markers

  • Utilize automated microscopy platforms

  • Apply machine learning for image analysis

These high-throughput approaches should incorporate appropriate controls and validation steps to address specificity concerns similar to those documented in the literature .

How might emerging antibody technologies enhance At2g33705 protein research?

Emerging technologies hold promise for advancing At2g33705 research:

• Single-domain antibodies (nanobodies):

  • Development of camelid-derived nanobodies against At2g33705

  • Enhanced penetration into plant tissues

  • Improved access to conformational epitopes

• Generative AI for antibody optimization:

  • Application of deep learning models to enhance antibody specificity

  • Computational affinity maturation as demonstrated in recent research

  • Zero-shot antibody design targeting specific At2g33705 epitopes

• Structural biology integration:

  • Determination of crystal structures of At2g33705 Antibody-antigen complexes

  • Analysis of binding conformations similar to approaches used in other antibody studies

  • Structure-guided epitope refinement

These approaches represent the cutting edge of antibody technology, integrating computational design with experimental validation .

What considerations should be made when implementing CRISPR-Cas9 studies in conjunction with At2g33705 Antibody research?

When combining CRISPR-Cas9 genome editing with antibody-based detection:

• Knockout validation strategy:

  • Design guide RNAs targeting At2g33705

  • Generate multiple independent knockout lines

  • Confirm editing by sequencing

  • Validate absence of protein using the At2g33705 Antibody

• Epitope tagging considerations:

  • Use CRISPR to introduce epitope tags (HA, FLAG, etc.)

  • Compare detection using At2g33705 Antibody vs. tag-specific antibodies

  • Assess whether tagging affects protein function

• Spatial and temporal expression studies:

  • Generate promoter-reporter fusions using CRISPR

  • Compare reporter expression with antibody-based detection

  • Create conditional knockout lines to study dynamic protein expression