BEE2 Antibody

What is the BEE2 protein target of this antibody?

BEE2 (brassinosteroid enhanced expression 2) is a basic helix-loop-helix (bHLH) transcription factor in Arabidopsis thaliana with UniProt accession number Q93VJ4. It functions as a transcriptional regulator involved in brassinosteroid signaling pathways, which are critical for various aspects of plant growth and development. BEE2 is part of a family of brassinosteroid early-response genes that mediate plant hormone responses and cell elongation processes .

What sample types can BEE2 antibody detect?

The BEE2 antibody (CSB-PA835947XA01DOA) is designed to detect the native BEE2 protein in Arabidopsis thaliana samples. It can be used with various plant tissue preparations including leaf extracts, seedling homogenates, root samples, and isolated nuclei fractions. The antibody is particularly useful for detecting BEE2 in molecular weight ranges corresponding to its predicted size (~27-30 kDa depending on post-translational modifications) .

What are the primary applications of BEE2 antibody in plant research?

BEE2 antibody is primarily employed in techniques such as Western blotting, immunoprecipitation (IP), chromatin immunoprecipitation (ChIP), and immunohistochemistry (IHC). These applications allow researchers to study BEE2 protein expression levels, localization patterns, protein-protein interactions, and DNA-binding activities within the context of brassinosteroid signaling and transcriptional regulation in Arabidopsis thaliana .

What are the optimal conditions for Western blotting with BEE2 antibody?

For optimal Western blot results with BEE2 antibody, researchers should consider the following protocol parameters:

• Sample preparation: Extract proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail.

• Gel electrophoresis: Use 10-12% SDS-PAGE gels for optimal resolution of BEE2 protein.

• Transfer conditions: Transfer proteins to PVDF membrane at 100V for 1 hour in standard Towbin buffer.

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute BEE2 antibody 1:1000 in blocking solution and incubate overnight at 4°C.

• Secondary antibody: Use anti-rabbit HRP-conjugated secondary antibody at 1:5000 dilution for 1 hour at room temperature.

• Detection: Develop using enhanced chemiluminescence (ECL) reagent with 1-5 minute exposure times.
  These conditions may require optimization based on specific laboratory equipment and sample characteristics.

How should I design immunoprecipitation experiments using BEE2 antibody?

For successful immunoprecipitation of BEE2 protein:

• Prepare fresh plant tissue lysate (approximately 500 μg total protein) in a non-denaturing lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, protease inhibitors).

• Pre-clear lysate with Protein A/G beads for 1 hour at 4°C to reduce non-specific binding.

• Incubate pre-cleared lysate with 2-5 μg of BEE2 antibody overnight at 4°C with gentle rotation.

• Add 30-50 μl of Protein A/G beads and incubate for 2-4 hours at 4°C.

• Wash beads 4-5 times with lysis buffer containing reduced detergent concentration (0.1-0.2%).

• Elute bound proteins by boiling in SDS sample buffer or using a specific elution buffer depending on downstream applications.

• Include appropriate controls: IgG isotype control and input sample (5-10% of initial lysate).
  This approach allows for the isolation of BEE2 protein complexes for further analysis by Western blotting or mass spectrometry.

How can I validate BEE2 antibody specificity in my experimental system?

Validating BEE2 antibody specificity is crucial for reliable research results. Consider these validation approaches:

• Genetic controls: Compare antibody detection in wild-type Arabidopsis versus bee2 knockout/knockdown lines.

• Peptide competition assay: Pre-incubate antibody with excess synthetic BEE2 peptide immunogen before application to samples. Specific signal should be blocked.

• Recombinant protein control: Test antibody against purified recombinant BEE2 protein and unrelated control proteins.

• Multiple antibody verification: When possible, compare results using independently generated antibodies against different epitopes of BEE2.

• Molecular weight verification: Confirm that detected bands match the predicted molecular weight of BEE2 (~27-30 kDa).

• RNA/protein correlation: Correlate antibody signal with mRNA expression levels across tissues or treatments.
  Thorough validation ensures that experimental observations can be confidently attributed to BEE2 protein.

What is the protocol for using BEE2 antibody in chromatin immunoprecipitation (ChIP) studies?

For ChIP experiments investigating BEE2 binding to genomic regions:

• Crosslink plant tissue with 1% formaldehyde for 10-15 minutes at room temperature.

• Quench crosslinking with 0.125 M glycine for 5 minutes.

• Isolate nuclei and sonicate chromatin to 200-500 bp fragments.

• Pre-clear chromatin with Protein A/G beads for 1-2 hours at 4°C.

• Incubate pre-cleared chromatin with 3-5 μg BEE2 antibody overnight at 4°C.

• Add Protein A/G beads and incubate for 2-3 hours at 4°C.

• Perform sequential washes with low salt, high salt, LiCl, and TE buffers.

• Elute DNA-protein complexes and reverse crosslinks (65°C overnight).

• Purify DNA using phenol-chloroform extraction or column-based methods.

• Analyze enriched DNA regions by qPCR or next-generation sequencing.
  This protocol enables identification of genomic regions directly bound by BEE2 transcription factor in vivo.

How can BEE2 antibody be used to study protein interactions in brassinosteroid signaling pathways?

To study BEE2 protein interactions in brassinosteroid signaling:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate BEE2 using the antibody as described in section 2.2

  • Analyze co-precipitated proteins by Western blotting using antibodies against suspected interacting partners

  • Alternatively, use mass spectrometry for unbiased identification of interaction partners

• Proximity-based labeling:

  • Generate BEE2-BioID or BEE2-TurboID fusion constructs

  • Express in plant cells and activate biotin labeling

  • Use BEE2 antibody to confirm proper expression and localization of fusion protein

  • Purify biotinylated proteins and identify by mass spectrometry

• Bimolecular Fluorescence Complementation (BiFC):

  • Use BEE2 antibody to validate expression levels of BiFC fusion constructs

  • Compare antibody-detected expression with fluorescence signal intensity
    These approaches can reveal direct and indirect protein interactions involving BEE2 in hormone response pathways.

What considerations are important for immunofluorescence microscopy with BEE2 antibody?

For successful immunofluorescence microscopy detecting BEE2 in plant tissues:

• Fixation: Fix plant tissue sections with 4% paraformaldehyde for 20-30 minutes, followed by permeabilization with 0.1-0.2% Triton X-100.

• Antigen retrieval: Consider gentle heat-mediated antigen retrieval in citrate buffer (pH 6.0) if initial staining is weak.

• Blocking: Use 5% normal goat serum in PBS with 0.1% Triton X-100 for 1-2 hours at room temperature.

• Primary antibody: Dilute BEE2 antibody 1:100 to 1:500 in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: Use fluorophore-conjugated anti-rabbit antibody (e.g., Alexa Fluor 488 or 594) at 1:500 dilution for 1-2 hours at room temperature.

• Counterstaining: DAPI (1 μg/ml) for nuclear visualization.

• Mounting: Use anti-fade mounting medium to prevent photobleaching.

• Controls: Include secondary-only controls and competitive peptide blocking controls to assess specificity.
  BEE2 should primarily show nuclear localization consistent with its role as a transcription factor, with potential cytoplasmic presence during certain developmental stages or treatments.

Why might I observe multiple bands when using BEE2 antibody in Western blots?

Multiple bands in BEE2 Western blots may occur for several research-relevant reasons:

• Post-translational modifications: BEE2 may undergo phosphorylation, ubiquitination, or other modifications that alter its molecular weight. Brassinosteroid signaling often involves phosphorylation cascades that could modify BEE2.

• Protein isoforms: Alternative splicing of BEE2 mRNA might generate protein variants of different sizes.

• Proteolytic processing: BEE2 might undergo regulated proteolysis as part of its functional cycle or during sample preparation.

• Cross-reactivity: The antibody might recognize other BEE family members (BEE1, BEE3) that share sequence homology with BEE2.
  To investigate these possibilities:

• Compare band patterns with predicted sizes of known isoforms

• Use phosphatase treatment to eliminate phosphorylation-dependent bands

• Include protease inhibitors during sample preparation

• Compare blots from wild-type and bee2 mutant plants to identify specific bands

How can I quantify BEE2 protein expression levels accurately?

For accurate quantification of BEE2 protein expression:

• Use appropriate loading controls:

  • For total protein normalization, consider Ponceau S staining or housekeeping proteins like actin or GAPDH

  • For nuclear protein normalization, use histone H3 or other nuclear markers

• Ensure linear detection range:

  • Perform a dilution series of your samples to confirm signal is within linear range

  • Use exposure times that avoid saturation of signal

• Replicate analysis:

  • Perform at least three biological replicates

  • Use technical replicates to assess method variability

• Standardization:

  • Include a standard sample across all blots for inter-blot comparisons

  • Consider using recombinant BEE2 protein as a quantitative standard

• Densitometry analysis:

  • Use software that allows background subtraction (ImageJ, Image Lab, etc.)

  • Define analysis parameters consistently across all samples

• Statistical analysis:

  • Apply appropriate statistical tests to determine significance of observed differences

  • Report means with standard deviation or standard error
    This methodical approach enhances reproducibility and reliability of BEE2 expression analysis.

What strategies help reduce high background when using BEE2 antibody?

To minimize background signal with BEE2 antibody:

• Blocking optimization:

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

  • Increase blocking time (2-3 hours at room temperature or overnight at 4°C)

• Antibody dilution:

  • Perform a dilution series to determine optimal antibody concentration

  • Consider using antibody diluents with background reducers

• Washing steps:

  • Increase number and duration of wash steps

  • Add low concentrations of detergent (0.05-0.1% Tween-20) to wash buffers

• Sample preparation:

  • Include additional clearing steps in sample preparation

  • Pre-absorb antibody with plant extract from bee2 mutant

• Incubation conditions:

  • Perform antibody incubations at 4°C rather than room temperature

  • Use gentle agitation during incubations and washes

• Secondary antibody optimization:

  • Titrate secondary antibody to minimize non-specific binding

  • Consider using highly cross-adsorbed secondary antibodies
    These approaches can significantly improve signal-to-noise ratio in BEE2 detection experiments.

How does BEE2 function compare with other BEE family proteins?

BEE2 belongs to a family of basic helix-loop-helix (bHLH) transcription factors that include BEE1 and BEE3 in Arabidopsis thaliana. Comparative analysis reveals:

• Expression patterns:

  • BEE2 shows strongest expression in elongating cells and developing tissues

  • BEE1 and BEE3 have partially overlapping but distinct expression domains

  • Triple bee1/bee2/bee3 mutants show stronger phenotypes than single mutants, indicating functional redundancy

• Hormone responsiveness:

  • All BEE proteins are rapidly induced by brassinosteroids

  • BEE2 also responds to auxin and gibberellin treatments

  • BEE proteins show differential responses to abscisic acid, with BEE2 being most strongly repressed

• Protein interactions:

  • BEE2 interacts with other bHLH proteins including IBH1 and PAR1

  • These interactions may differ among BEE family members, contributing to their specific functions

  • BEE2 shows stronger interaction with components of the brassinosteroid signaling pathway

• Target genes:

  • ChIP studies suggest both common and distinct genomic targets among BEE proteins

  • BEE2 appears to have greater affinity for certain E-box variants in promoter regions
    Understanding these differences is crucial when interpreting results from BEE2 antibody studies in the broader context of brassinosteroid signaling.

What are the key experimental considerations when studying BEE2 in different plant tissues?

When investigating BEE2 across different plant tissues:

• Developmental timing:

  • BEE2 expression varies significantly during development

  • Standardize sample collection by developmental stage rather than chronological age

• Tissue-specific extraction protocols:

  • Optimize extraction buffers for different tissues (roots vs. leaves vs. flowers)

  • Consider tissue-specific interfering compounds that may affect antibody binding

• Reference selection:

  • Use tissue-appropriate reference genes/proteins

  • Consider normalized protein loading based on fresh weight for cross-tissue comparisons

• Subcellular localization:

  • BEE2 nuclear localization may vary by tissue and developmental stage

  • Compare nuclear enrichment protocols for different tissue types

• Environmental conditions:

  • Standardize growth conditions as BEE2 is environmentally responsive

  • Document light conditions, temperature, and humidity as these affect brassinosteroid signaling

• Hormone status:

  • Consider endogenous hormone levels in different tissues

  • Pre-treatment with brassinazole (brassinosteroid biosynthesis inhibitor) can help normalize baseline levels
    These considerations ensure meaningful comparisons of BEE2 expression and function across tissue types.

How conserved is BEE2 across different plant species?

BEE2 conservation analysis reveals important evolutionary and functional insights:

• Epitope conservation: The BEE2 antibody raised against Arabidopsis protein may cross-react with homologs from closely related species (particularly Brassicaceae), but validation is essential.

• Functional conservation: Despite sequence divergence, BEE2 homologs appear to maintain their core function in brassinosteroid response across species, though with potential regulatory differences.

• Cross-species applications: Researchers studying BEE2 in non-Arabidopsis species should perform rigorous validation of antibody specificity through Western blotting and immunoprecipitation before proceeding with experiments.

• Evolutionary insights: Regions with highest conservation across species likely represent functionally critical domains that could be targeted for specific antibody development. These comparative analyses provide context for extrapolating findings from Arabidopsis BEE2 studies to other plant systems.