ATHB-16 Antibody

What is ATHB-16 and why is it significant in plant research?

ATHB-16 is a homeodomain protein belonging to the plant-specific HD-Zip class of transcriptional regulators in Arabidopsis thaliana. It functions as a negative regulator of the ABA signal pathway, acting downstream of the protein phosphatase ABI1 . The protein is particularly significant because it represents a critical link between the protein phosphatase 2C (PP2C) activity of ABI1 and gene regulation in plants. Studies have demonstrated that ATHB-16 gene expression is upregulated by ABA and during drought stress conditions, indicating its important role in plant stress responses .

What applications has the ATHB-16 antibody been validated for?

The ATHB-16 antibody (e.g., product code CSB-PA839077XA01DOA) has been tested and validated for several research applications including:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western blotting (WB) for identification of ATHB-16 antigen

These applications make the antibody suitable for detecting native and recombinant ATHB-16 protein in experimental settings focusing on plant stress responses and hormonal signaling pathways.

What is the optimal storage condition for ATHB-16 antibody?

Upon receipt, ATHB-16 antibody should be stored at -20°C or -80°C to maintain its activity. Repeated freeze-thaw cycles should be avoided to prevent degradation of the antibody . The antibody is typically provided in a storage buffer containing 50% glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . Once thawed for use, the antibody should be kept at 4°C for short-term use and returned to -20°C for long-term storage.

How should researchers optimize Western blot protocols for ATHB-16 detection?

For optimal Western blot detection of ATHB-16:

• Sample preparation: Extract plant tissue in a buffer containing protease inhibitors to prevent degradation of ATHB-16 protein.

• Gel electrophoresis: Use 10-12% SDS-PAGE gels for good resolution of ATHB-16.

• Transfer: Transfer proteins to PVDF or nitrocellulose membranes at 100V for 60-90 minutes.

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

• Primary antibody: Dilute ATHB-16 antibody 1:2000 to 1:5000 in blocking buffer and incubate overnight at 4°C .

• Secondary antibody: Use anti-rabbit IgG-HRP at recommended dilution.

• Detection: Visualize using enhanced chemiluminescence (ECL) reagents.

Include positive controls from Arabidopsis thaliana tissues known to express ATHB-16, especially those treated with ABA, as expression levels increase significantly after ABA treatment .

What controls should be included when working with ATHB-16 antibody?

When designing experiments with ATHB-16 antibody, include:

• Positive control: Arabidopsis thaliana tissue samples with confirmed ATHB-16 expression, particularly those treated with ABA to enhance expression.

• Negative control: Samples from tissues with minimal ATHB-16 expression or tissues from knockout plants.

• Antibody control: Primary antibody omission to check for non-specific binding of secondary antibody.

• Loading control: Anti-AtpB antibody or other housekeeping proteins to ensure equal sample loading .

• Peptide competition: Pre-incubation of antibody with immunizing peptide to confirm specificity.

These controls help validate experimental results and ensure the specificity of the antibody-antigen interaction.

How can ATHB-16 antibody be used to study ABA signaling pathways?

To investigate ABA signaling using ATHB-16 antibody:

• Protein interaction studies: Co-immunoprecipitation (Co-IP) experiments can identify interactions between ATHB-16 and other proteins, particularly ABI1. Research has shown that ATHB-16 physically interacts with ABI1, and this interaction is dependent on the PP2C activity of ABI1's catalytic domain .

• Chromatin immunoprecipitation (ChIP): Use ATHB-16 antibody to identify DNA binding sites and target genes of ATHB-16 in the context of ABA responses.

• Phosphorylation analysis: Since ATHB-16 contains a predicted phosphorylation site at serine residue S67 in the consensus motif KRRLSINQV adjacent to the HD α-helix 1, researchers can use the antibody in combination with phospho-specific antibodies to study how phosphorylation affects ATHB-16 function .

• Transcriptional regulation studies: Analyze how ATHB-16 affects reporter gene expression using constructs with promoters containing ATHB-16 binding sites. In protoplast systems, ATHB-16 has shown to increase reporter expression 7-fold, with a further increase to 17-fold in the presence of ABA .

What is known about the ATHB-16 interaction with ABI1 and how can it be studied?

ATHB-16 interaction with ABI1 can be studied through:

Research has shown that the interaction between ATHB-16 and ABI1 positively correlates with the PP2C activity of the ABI1 catalytic domain and is abolished in catalytically inactive ABI1 mutants .

How does ATHB-16 regulate its own expression and how can this be measured?

ATHB-16 appears to regulate its own promoter activity, potentially creating a feedback loop in ABA responses. This can be studied using:

• Reporter gene assays: Transgenic Arabidopsis expressing luciferase under the control of the ATHB-16 promoter shows remarkable ABA-dependent reporter activation, with induction levels beyond 2000-fold. Maximum expression levels (1.7 × 10^4 LU/μg) exceed those of the strong 35S promoter (1.3 × 10^4 LU/μg) .

• Time-course experiments: Reporter activation becomes detectable within 4 hours after ABA addition (10 μM), reaching half-maximal levels after approximately 12 hours .

• Western blot analysis: Use ATHB-16 antibody to quantify protein levels following ABA treatment or in different genetic backgrounds (e.g., abi1 mutant).

• qRT-PCR: Measure ATHB-16 transcript levels in parallel with protein detection to correlate transcriptional and translational regulation.

What are common issues when using ATHB-16 antibody and how can they be resolved?

How can researchers verify the specificity of ATHB-16 antibody?

To verify antibody specificity:

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples. Signal disappearance confirms specificity.

• Knockout/knockdown validation: Compare antibody reactivity in wild-type versus ATHB-16 knockout or knockdown plants.

• Overexpression systems: Test detection in systems with ATHB-16 overexpression to confirm signal increase correlates with protein level.

• Multiple antibody comparison: If available, use different antibodies targeting different epitopes of ATHB-16.

• Mass spectrometry: Confirm the identity of immunoprecipitated proteins using mass spectrometry.

How is ATHB-16 involved in plant stress responses beyond ABA signaling?

Recent research indicates that ATHB-16, as a transcriptional regulator in the HD-Zip class, plays broader roles in plant stress adaptation:

• Drought response: ATHB-16 gene expression is upregulated during drought stress, independent of its ABA-mediated regulation .

• Transcriptional network: ATHB-16 may function as a master switch for ABA-specific developmental adaptations, potentially regulating multiple downstream genes.

• Self-regulation: The remarkable ABA-dependent upregulation of ATHB-16 promoter activity (>2000-fold induction) suggests a significant amplification mechanism in stress response pathways .

What methods are used to study ATHB-16 DNA binding properties?

To investigate ATHB-16 DNA binding:

• Electrophoretic mobility shift assay (EMSA): Determine direct binding of purified ATHB-16 to DNA sequences.

• Chromatin immunoprecipitation (ChIP): Use ATHB-16 antibody to identify genomic binding sites in vivo.

• Reporter gene assays: Study promoter activation using ATHB-16 binding sites. Research has shown that point mutations in ATHB-16 binding sites (CAATTGTTA) abolish reporter activation, confirming sequence-specific binding .

• Deletion analysis: Study how removal of DNA-binding domains (e.g., deletion of HD α-helix 3) affects ATHB-16 function. Such deletions have been shown to result in only background levels of reporter expression .

How can ATHB-16 antibody be used in comparative studies across plant species?

While ATHB-16 antibody is specifically raised against Arabidopsis thaliana ATHB-16, comparative studies can be designed to:

• Identify homologs: Use sequence alignment to identify ATHB-16 homologs in other plant species.

• Cross-reactivity testing: Evaluate antibody cross-reactivity with homologous proteins from related plant species.

• Conservation analysis: Compare ATHB-16 function across species by combining antibody-based detection with functional assays.

• Evolutionary studies: Investigate the conservation of ABA signaling pathways across plant lineages by examining ATHB-16-like proteins and their interactions.

The approach would be similar to the cross-species reactivity demonstrated by antibodies like Anti-AtpB, which shows reactivity across diverse plant species including Arabidopsis thaliana, Hordeum vulgare, Glycine max, and Oryza sp. .