BON2 Antibody

What is BON2 Antibody and what is its target in plant research?

BON2 Antibody is a polyclonal antibody raised in rabbits that specifically targets the BON2 protein (UniProt Q5S1W2) in Arabidopsis thaliana (Mouse-ear cress). BON2 (BONZAI 2) belongs to a family of proteins involved in plant growth regulation and response to environmental stresses .
The antibody is generated using recombinant Arabidopsis thaliana BON2 protein as the immunogen and is purified using antigen affinity chromatography. This polyclonal IgG antibody is primarily used for detecting endogenous BON2 protein expression in plant samples .

What validated applications exist for BON2 Antibody?

BON2 Antibody has been validated for the following applications:

How should antibody specificity be validated when working with BON2 Antibody?

Antibody specificity is critical for reliable research outcomes. For BON2 Antibody, a comprehensive validation approach should include:

• Positive and negative controls: Test samples from wild-type and BON2 knockout Arabidopsis lines.

• Peptide competition assay: Pre-incubate BON2 Antibody with excess purified BON2 protein to confirm signal reduction.

• Cross-reactivity assessment: Test against related proteins (e.g., BON1, BON3) to verify specificity.

• Multiple detection methods: Compare results across different techniques (WB, ELISA, immunofluorescence).

• Signal localization: Verify that observed signals match expected subcellular localization patterns.
  This approach aligns with the broader principles of antibody validation for research applications, where verification of specificity is crucial for experimental integrity .

What considerations are important when using BON2 Antibody in different plant model systems?

When extending BON2 Antibody use beyond Arabidopsis thaliana:

What is the optimal protocol for Western blot using BON2 Antibody?

The following optimized protocol incorporates best practices for detecting BON2 protein:
Sample Preparation:

• Extract total protein using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitor cocktail

• Use 20-50μg total protein per lane
  SDS-PAGE and Transfer:

• Separate proteins on 10-12% polyacrylamide gel

• Transfer to PVDF membrane (0.45μm) at 100V for 60 minutes or 30V overnight
  Immunoblotting:

• Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Incubate with BON2 Antibody (1:1000 dilution) in 5% BSA/TBST overnight at 4°C

• Wash 3 times with TBST, 10 minutes each

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

• Wash 3 times with TBST, 10 minutes each

• Develop using ECL substrate and detect signal
  Critical Controls:

• Include wild-type and BON2 knockout samples

• Use recombinant BON2 protein as positive control

• Include loading control (e.g., anti-actin antibody)
  Expected band size for BON2 protein is approximately 50-55 kDa .

How should BON2 Antibody be applied in ELISA experiments?

For successful ELISA with BON2 Antibody, follow this methodology:
Indirect ELISA Protocol:

• Coat 96-well plate with 100μL of plant extract (1-10μg protein) in coating buffer (50mM carbonate-bicarbonate, pH 9.6) overnight at 4°C

• Wash 3 times with PBST

• Block with 200μL 3% BSA in PBST for 2 hours at room temperature

• Wash 3 times with PBST

• Add 100μL BON2 Antibody (1:2000 dilution) in 1% BSA/PBST for 2 hours at room temperature

• Wash 5 times with PBST

• Add 100μL HRP-conjugated anti-rabbit antibody (1:5000) for 1 hour at room temperature

• Wash 5 times with PBST

• Add 100μL TMB substrate and incubate for 15-30 minutes

• Stop reaction with 50μL 2N H₂SO₄

• Read absorbance at 450nm
  Sandwich ELISA (for increased specificity):

• Coat plate with capture antibody (e.g., commercially available BON2 antibody from different host)

• Add sample containing BON2 protein

• Detect with BON2 Antibody followed by appropriate secondary antibody
  This approach allows for quantitative analysis of BON2 protein levels across different experimental conditions .

What are common issues when using BON2 Antibody and how can they be resolved?

How can advanced techniques enhance research using BON2 Antibody?

Combining Immunoprecipitation with Mass Spectrometry:

• Use BON2 Antibody to immunoprecipitate BON2 and associated proteins

• Analyze protein complexes via mass spectrometry to identify interaction partners

• Validate interactions using reciprocal co-immunoprecipitation
  Chromatin Immunoprecipitation (ChIP) Applications:
  If BON2 has DNA-binding capabilities or associates with chromatin, ChIP-seq can identify genomic binding sites:

• Cross-link protein-DNA complexes in plant tissue

• Immunoprecipitate with BON2 Antibody

• Reverse cross-linking and sequence DNA fragments
  Proximity Ligation Assay:
  For studying protein-protein interactions in situ:

• Use BON2 Antibody alongside antibody against putative interaction partner

• Apply secondary antibodies with oligonucleotide probes

• Ligate and amplify signal when proteins are in close proximity
  These advanced applications should be preceded by thorough antibody validation specific to each technique .

How can computational approaches complement BON2 Antibody-based research?

Recent advances in computational biology offer powerful complementary approaches to antibody-based research:

• Epitope prediction: Computational tools can identify likely epitopes recognized by BON2 Antibody, helping researchers understand potential cross-reactivity with related proteins.

• Structure-based analysis: As noted in recent studies on antibody design, "a fine-tuned RFdiffusion network is capable of designing de novo antibody variable heavy chains (VHH's) that bind user-specified epitopes" . Similar approaches could be applied to predict BON2-antibody interactions.

• Custom specificity profiles: Building on emerging techniques in the field, researchers can potentially design "novel antibody sequences with customized specificity profiles" . This approach could be valuable for developing more specific versions of BON2 Antibody.

• Integration with proteomics data: Combining antibody-based detection with proteomics datasets can provide comprehensive understanding of BON2 function in different contexts.
  These computational approaches can enhance traditional antibody-based research by providing additional layers of validation and interpretation .

What considerations are important when developing phospho-specific BON2 antibodies?

If BON2 undergoes phosphorylation as part of its regulatory mechanism, developing phospho-specific antibodies requires special considerations:

• Identification of phosphorylation sites: Mass spectrometry analysis should be conducted to identify specific phosphorylation sites on BON2 protein.

• Peptide design: For antibody generation, synthetic phosphopeptides containing the phosphorylation site of interest plus 5-10 flanking amino acids should be used as immunogens.

• Dual validation approach: As described in phosphoprotein-based biomarker research, validation should include "phosphorylation state-specific antibodies... for detection" alongside verification with phosphatase treatments.

• Application-specific controls: Each application requires specific controls, such as phosphatase-treated samples and phospho-mimetic mutants (S/T→D/E).
  This approach builds on established methodologies for developing and validating phospho-specific antibodies for research applications .

What are the best practices for quantifying and reporting BON2 Antibody experimental results?

For rigorous research with BON2 Antibody, follow these quantification and reporting guidelines:
Western Blot Quantification:

• Use biological replicates (n≥3) and technical replicates (n≥2)

• Normalize BON2 signal to appropriate loading controls (actin, tubulin, or total protein)

• Use digital image analysis software for densitometry

• Report mean values with standard error/deviation

• Include representative blot images showing full molecular weight range
  ELISA Data Reporting:

• Include standard curve with known quantities of recombinant BON2

• Report both raw OD values and calculated concentrations

• Include detection limits and linear range of assay

• Report coefficient of variation between replicates
  Statistical Analysis:

• Clearly state statistical tests used and why they're appropriate

• Report exact p-values rather than thresholds

• Include power analysis to justify sample size

• Consider multiple testing corrections when applicable
  These practices ensure reproducibility and align with standards for antibody-based research reporting .