ATHB-53 Antibody

What is ATHB-53 antibody and what are its primary research applications?

ATHB-53 antibody is a rabbit polyclonal antibody generated against the Arabidopsis thaliana ATHB-53 protein (also known as Homeobox-8). This antibody is primarily used for detecting ATHB-53 protein expression in plant tissues, particularly Arabidopsis . The antibody has validated applications in several experimental techniques including Western Blotting (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) .

The antibody recognizes the ATHB-53 protein, which belongs to the homeodomain-leucine zipper family of transcription factors in plants. For optimal research applications, ATHB-53 antibody should be stored at -20°C to -80°C to maintain its binding efficacy and specificity .

How should researchers validate ATHB-53 antibody specificity before experimental use?

Validation of ATHB-53 antibody specificity is a critical step before conducting any experiment. The methodological approach should include:

• Positive control testing: Use the recombinant immunogen protein provided with the antibody (200μg) as a positive control in Western blot analysis .

• Pre-immune serum comparison: Compare results with the pre-immune serum (provided in the antibody package) to identify potential non-specific binding .

• Cross-reactivity assessment: Though not explicitly stated for ATHB-53, cross-reactivity testing follows standard practices similar to other antibodies where testing against related proteins (other ATHB family members) is recommended .

• Knockout/knockdown validation: When possible, use tissue samples from ATHB-53 knockout or knockdown plants as negative controls.

This multi-step validation ensures experimental rigor and prevents misinterpretation of results in subsequent experiments.

What are the optimal conditions for using ATHB-53 antibody in Western blot applications?

When using ATHB-53 antibody for Western blot applications, researchers should follow these methodological guidelines for optimal results:

• Sample preparation:

  • Extract proteins from plant tissues using a buffer containing phosphatase and protease inhibitors

  • Load 20-50μg of total protein per lane

• Dilution optimization:

  • Start with a 1:1000 dilution in blocking buffer (typically 5% non-fat dry milk in TBST)

  • Adjust based on signal intensity after initial experiments

• Detection system:

  • Use HRP-conjugated anti-rabbit secondary antibody (typically 1:5000 dilution)

  • Develop using enhanced chemiluminescence (ECL) substrate

• Incubation conditions:

  • Primary antibody: Overnight at 4°C with gentle rocking

  • Secondary antibody: 1 hour at room temperature

The antibody is formulated in a preservative solution containing 0.03% Proclin 300 with 50% Glycerol in 0.01M PBS at pH 7.4, which should be considered when planning dilution strategies .

How can researchers troubleshoot weak or non-specific signals when using ATHB-53 antibody?

When encountering weak or non-specific signals with ATHB-53 antibody, researchers should implement the following troubleshooting strategy:

For research involving plant samples, note that high levels of phenolic compounds and polysaccharides may interfere with antibody binding, requiring specialized extraction protocols to improve signal quality.

How does ATHB-53 antibody compare to antibodies against other plant transcription factors for chromatin immunoprecipitation (ChIP) experiments?

When considering ATHB-53 antibody for ChIP applications, researchers should be aware of several important methodological considerations compared to antibodies targeting other plant transcription factors:

• Fixation protocol optimization: ATHB-53 typically requires standard formaldehyde fixation (1% for 10 minutes), similar to protocols used for other plant homeodomain proteins.

• Chromatin fragmentation: For ATHB-53 ChIP, sonication conditions should be optimized to generate DNA fragments of 200-500bp, which is critical for successful immunoprecipitation of ATHB-53-bound chromatin regions.

• IP efficiency comparison: While specific data for ATHB-53 antibody IP efficiency is limited, researchers should expect performance in the range typical for plant transcription factor antibodies (5-15% pulldown of target loci).

• Antibody selection criteria: For ChIP applications, using antigen-affinity purified antibodies like the ATHB-53 polyclonal provides better specificity than crude sera, but monoclonal antibodies (not yet widely available for ATHB-53) often provide more consistent results across experiments.

Since the ATHB-53 antibody is polyclonal and purified by antigen affinity , it has potential for ChIP applications, though researchers may need to optimize protocols specifically for this antibody.

What considerations should be taken into account when using ATHB-53 antibody for co-immunoprecipitation studies of plant transcription factor complexes?

When using ATHB-53 antibody for co-immunoprecipitation (co-IP) of transcription factor complexes, researchers should address these critical methodological considerations:

• Buffer composition:

  • Use low-stringency buffers (150mM NaCl, 0.5% NP-40) initially to preserve protein-protein interactions

  • Add specific components to maintain plant transcription factor complex integrity:

    • 10% glycerol to stabilize protein interactions

    • 1mM DTT to maintain reduced cysteines

    • Appropriate protease inhibitor cocktail

• Cross-linking considerations:

  • For transient interactions, consider mild crosslinking (0.1-0.5% formaldehyde, 5-10 minutes)

  • For stable complexes, direct co-IP without crosslinking may be sufficient

• Antibody orientation options:

  • Forward IP: Use ATHB-53 antibody to pull down complexes and detect partner proteins

  • Reverse IP: Use antibodies against suspected interaction partners to detect ATHB-53

• Controls:

  • Pre-immune serum control (supplied with the antibody)

  • IgG isotype control

  • Input sample (5-10% of starting material)

  • Ideally, ATHB-53 knockout/knockdown negative controls

The antibody's format (liquid formulation in glycerol/PBS) needs to be considered when calculating the final buffer composition for immunoprecipitation reactions.

How should researchers design experiments to study ATHB-53 expression patterns across different plant developmental stages?

When studying ATHB-53 expression patterns across developmental stages, researchers should implement a comprehensive experimental design:

• Tissue sampling strategy:

  • Collect tissues from key developmental stages (seedling, vegetative growth, flowering, seed development)

  • Include multiple tissue types (roots, shoots, leaves, flowers, siliques)

  • Use biological replicates (minimum n=3) for statistical validity

• Protein extraction optimization:

  • Use a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, protease inhibitors

  • Adjust extraction procedures based on tissue type (higher detergent concentrations for more fibrous tissues)

• Detection methods:

  • Western blot using ATHB-53 antibody (1:1000 dilution)

  • Immunohistochemistry for spatial localization

  • Consider parallel qRT-PCR for transcript level correlation

• Quantification approach:

  • Normalize ATHB-53 protein levels to appropriate loading controls (actin, tubulin, or total protein)

  • Present data as relative expression compared to a reference stage or tissue

  • Include statistical analysis (ANOVA with post-hoc tests) to identify significant differences

This design enables researchers to generate a comprehensive spatiotemporal expression map of ATHB-53 protein across development, providing insights into its regulatory roles.

What are the most effective methods for analyzing post-translational modifications of ATHB-53 using the antibody?

To effectively analyze post-translational modifications (PTMs) of ATHB-53 protein, researchers should employ a systematic multi-method approach:

• Phosphorylation analysis:

  • Compare standard and Phos-tag™ SDS-PAGE gels for mobility shift detection

  • Perform immunoprecipitation with ATHB-53 antibody followed by phospho-specific staining

  • Treatment groups should include:

    • Control conditions

    • Phosphatase treatment (to remove phosphorylation)

    • Stress conditions that may induce phosphorylation

• Ubiquitination detection:

  • Immunoprecipitate with ATHB-53 antibody and probe with anti-ubiquitin antibodies

  • Add proteasome inhibitors (MG132) to cell extracts to stabilize ubiquitinated forms

  • Compare protein half-life under different conditions

• SUMOylation assessment:

  • Co-immunoprecipitation with ATHB-53 antibody followed by anti-SUMO probing

  • Analyze molecular weight shifts corresponding to SUMO addition

  • Validate with SUMO protease treatments

• Technical considerations:

  • Include appropriate controls (untreated lysates, in vitro modified recombinant protein)

  • Optimize extraction conditions to preserve labile PTMs (use phosphatase inhibitors, deubiquitinase inhibitors)

  • Consider mass spectrometry validation of identified PTMs after immunoprecipitation

This comprehensive approach allows for detailed characterization of ATHB-53 PTMs and their functional significance in different physiological contexts.

How can ATHB-53 antibody be applied to investigate drought response mechanisms in plants?

For investigating ATHB-53's role in drought response mechanisms, researchers should implement the following methodological approach:

• Experimental design for drought studies:

  • Establish controlled drought conditions (soil moisture monitoring, withholding water, or PEG treatment)

  • Define sampling timepoints (early response: 0-6h; intermediate: 24-48h; late: 3-7d)

  • Include appropriate controls (well-watered plants, known drought-responsive genes)

• Protein expression analysis:

  • Extract proteins from drought-stressed and control plants

  • Analyze ATHB-53 protein levels by Western blotting using the antibody (1:1000 dilution)

  • Quantify expression changes relative to drought severity and duration

• Subcellular localization studies:

  • Perform cell fractionation followed by Western blotting

  • Alternatively, use immunohistochemistry on fixed tissue sections

  • Track potential nuclear-cytoplasmic shuttling upon stress

• Protein-protein interaction analysis:

  • Conduct co-immunoprecipitation using ATHB-53 antibody to identify stress-specific interacting partners

  • Compare interaction profiles between normal and drought conditions

This approach enables comprehensive characterization of ATHB-53's involvement in drought response pathways, potentially identifying novel regulatory mechanisms in plant stress adaptation.

What considerations should be taken into account when analyzing conflicting ATHB-53 expression data between transcript and protein levels?

When encountering discrepancies between ATHB-53 transcript and protein expression data, researchers should consider these methodological approaches to resolve conflicts:

• Time-course analysis:

  • Implement high-resolution time-course experiments to detect temporal delays between mRNA and protein expression

  • Typical sampling intervals: 0, 2, 4, 8, 12, 24, 48 hours following treatment or developmental trigger

• Post-transcriptional regulation assessment:

  • Analyze mRNA stability using actinomycin D treatment (transcription inhibitor)

  • Examine polysome association of ATHB-53 mRNA to assess translation efficiency

  • Investigate potential regulatory roles of miRNAs targeting ATHB-53 transcripts

• Protein turnover evaluation:

  • Conduct cycloheximide chase experiments to determine ATHB-53 protein half-life

  • Compare protein stability under different conditions where discrepancies are observed

  • Analyze potential involvement of ubiquitin-proteasome degradation using proteasome inhibitors

• Technical validation:

  • Confirm antibody specificity using the provided controls

  • Validate qRT-PCR primers for specificity to ATHB-53 vs. related homeobox genes

  • Use multiple reference genes/proteins for normalization

This systematic approach helps distinguish between transcriptional, post-transcriptional, and post-translational regulatory mechanisms affecting ATHB-53 expression.

How do different fixation and permeabilization methods affect ATHB-53 antibody performance in immunohistochemistry of plant tissues?

When conducting immunohistochemistry (IHC) with ATHB-53 antibody in plant tissues, the fixation and permeabilization methods significantly impact results:

• Comparison of fixation methods:

  • Paraformaldehyde (4%): Provides good morphology preservation while maintaining ATHB-53 epitope accessibility; recommended as primary fixation method

  • Glutaraldehyde (0.5-2%): Stronger fixation but may mask epitopes; recommended only when studying specific subcellular structures

  • Methanol-acetone: Less optimal for ATHB-53 detection but provides better preservation of some post-translational modifications

  • Non-aldehyde ZnSO₄ fixation: Alternative for preserving certain phospho-epitopes

• Permeabilization optimization:

  • Triton X-100 (0.1-0.5%): Standard permeabilization for most plant tissues

  • Enzymatic digestion: Limited cell wall digestion (1% cellulase, 0.5% macerozyme) may improve antibody penetration in thicker sections

  • Freeze-thaw cycles: Alternative method that can improve antibody access to nuclear ATHB-53

• Tissue-specific considerations:

  • Young tissues: Require gentler permeabilization (0.1% Triton X-100)

  • Mature leaves: May require longer permeabilization or higher detergent concentration

  • Reproductive tissues: Often need specialized embedding methods to preserve structure while allowing antibody penetration

For optimal results with ATHB-53 antibody in IHC applications, a recommended starting protocol would include 4% paraformaldehyde fixation for 4 hours followed by 0.3% Triton X-100 permeabilization for 30 minutes, with antibody dilution at 1:500 .

What are the critical differences in experimental design when using ATHB-53 antibody for research in crop species versus model organisms like Arabidopsis?

When adapting ATHB-53 antibody research from Arabidopsis to crop species, researchers should consider these critical experimental design modifications:

• Antibody cross-reactivity validation:

  • Perform initial Western blot using recombinant homologs from target crop species

  • Compare protein sequences of ATHB-53 orthologs to assess epitope conservation

  • Consider antibody affinity purification against crop-specific protein for improved specificity

• Extraction protocol adaptations:

  • Cereal crops: Increase reducing agent concentration (5-10mM DTT) to manage higher disulfide content

  • Legumes: Add PVPP (2-5%) to remove interfering phenolic compounds

  • Solanaceous crops: Include specific protease inhibitors targeting abundant proteases in these species

• Tissue processing differences:

  • Adjust tissue:buffer ratios based on water content and cell wall composition of crop tissues

  • Consider tissue-specific homogenization methods (higher speeds/longer times for fibrous crop tissues)

  • Implement additional clarification steps (centrifugation at 20,000g for 20 minutes) for crop extracts

• Experimental controls:

  • Include tissues from multiple developmental stages to identify temporal expression patterns

  • Use phylogenetically distant tissues as negative controls

  • If available, include RNAi or CRISPR-modified lines with reduced target expression

• Signal detection optimization:

  • Often require longer exposure times for crop species due to potentially lower affinity

  • Consider signal amplification methods (biotin-streptavidin systems) for weak signals

  • Adjust blocking conditions (5% milk may be insufficient; consider 3% BSA or commercial blocking reagents)

These adaptations address the challenges of applying antibodies developed against model organism proteins to more complex and diverse crop species.