AUG8 Antibody

What is the AUG8 protein in Arabidopsis thaliana and what cellular processes is it involved in?

AUG8 (Q9SUH5) in Arabidopsis thaliana belongs to the augmin complex family of proteins that play crucial roles in microtubule organization during cell division. The protein is involved in nucleation of microtubules from existing microtubules, contributing to spindle formation and phragmoplast organization during mitosis and cytokinesis. Researchers typically use AUG8 antibodies to study microtubule dynamics, particularly during plant cell division processes where spindle organization and integrity are critical .

What are the recommended techniques for validating AUG8 antibody specificity in Arabidopsis samples?

Validation of AUG8 antibody specificity should follow a multi-step approach:

• Western blot analysis using wild-type Arabidopsis and aug8 mutant lines

• Immunoprecipitation followed by mass spectrometry

• Immunolocalization studies comparing wild-type vs. aug8 knockout tissues

• Peptide competition assays using the immunizing peptide

Always include appropriate positive and negative controls in each experiment. For negative controls, aug8 mutant lines or tissues where AUG8 expression is naturally absent should be used, while recombinant AUG8 protein can serve as a positive control .

Which experimental techniques are most suitable for studying AUG8 localization during different cell cycle phases?

For studying AUG8 localization across cell cycle phases, researchers should consider:

• Immunofluorescence microscopy with cell cycle markers (DAPI for DNA, cyclin antibodies)

• Live-cell imaging using GFP-tagged AUG8 in stable transformants

• Super-resolution microscopy (SIM or STED) for detailed subcellular localization

• Co-immunoprecipitation with cell cycle-specific proteins

For optimal results, synchronize Arabidopsis cell cultures using aphidicolin (G1/S block) or propyzamide (metaphase block) before fixation and immunostaining with the AUG8 antibody .

How should researchers design experiments to investigate AUG8 interactions with other augmin complex components?

When investigating AUG8 interactions with other augmin components:

• Co-immunoprecipitation protocol: Use AUG8 antibody (CSB-PA154725XA01DOA) immobilized on protein A/G magnetic beads in extraction buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitors). Incubate with plant extracts for 2-3 hours at 4°C, wash 4-5 times, and analyze by Western blot with antibodies against other augmin subunits.

• Proximity-dependent biotin identification (BioID): Generate transgenic Arabidopsis expressing AUG8-BioID fusions, treat with biotin, and identify interacting proteins by streptavidin pulldown followed by mass spectrometry.

• Yeast two-hybrid screening: Use AUG8 as bait against an Arabidopsis cDNA library, focusing on identification of novel interactions.

• Fluorescence resonance energy transfer (FRET): Express AUG8-CFP and potential interactors tagged with YFP to quantify in vivo protein-protein interactions in plant cells .

What are the optimal parameters for immunohistochemistry using AUG8 antibody in different plant tissues?

Optimal parameters for AUG8 immunohistochemistry vary by tissue type:

For all tissues, include permeabilization with 0.2% Triton X-100 for 15 minutes after fixation and blocking with 3% BSA in PBS for 1 hour before antibody incubation. For negative controls, use pre-immune serum at the same concentration as the primary antibody .

How can researchers effectively use AUG8 antibody in chromatin immunoprecipitation (ChIP) experiments?

For ChIP experiments using AUG8 antibody:

• Crosslinking: Fix plant tissue with 1% formaldehyde for 10 minutes under vacuum, quench with 0.125M glycine.

• Chromatin preparation: Extract and shear chromatin to 200-500bp fragments using sonication (10-15 cycles of 30 seconds on/30 seconds off).

• Immunoprecipitation: Pre-clear chromatin with protein A/G beads, then incubate with AUG8 antibody (2-5μg) overnight at 4°C. Add protein A/G beads for 2-3 hours.

• Washes and elution: Perform stringent washing series (low salt, high salt, LiCl, and TE buffers). Elute with 1% SDS, 0.1M NaHCO₃ buffer at 65°C.

• Reverse crosslinking and purification: Treat with proteinase K, reverse crosslink at 65°C overnight, purify DNA using phenol-chloroform extraction.

• Analysis: Perform qPCR on regions of interest or submit for next-generation sequencing.

Optimize antibody concentration using a titration series (1-10μg per reaction) to determine minimal effective concentration for specific enrichment .

What are common causes of false positives/negatives when using AUG8 antibody, and how can researchers address them?

Common causes of false results with AUG8 antibody include:

False Positives:

• Cross-reactivity with related proteins: Perform pre-absorption with recombinant related proteins

• Non-specific binding to denatured proteins: Add 0.1% SDS to antibody dilution buffer

• Insufficient blocking: Extend blocking time to 2 hours with 5% BSA or milk

False Negatives:

• Epitope masking: Try multiple antigen retrieval methods (heat, enzymatic, high pH)

• Antibody concentration too low: Optimize with titration series

• Protein degradation: Add protease inhibitors freshly to all buffers

• Overfixation: Reduce fixation time or try different fixatives

To validate results, always:

• Include appropriate controls (positive, negative, secondary-only)

• Confirm specificity with multiple detection methods

• Validate with genetic approaches (aug8 mutants, RNAi lines)

How should researchers interpret contradictory data between AUG8 antibody results and transcriptome data?

When facing contradictions between AUG8 antibody results and transcriptome data:

• Establish timeline: Protein expression often lags behind transcript levels, so consider temporal factors.

• Methodologically validate: Confirm antibody specificity using western blot against recombinant AUG8 protein and aug8 knockout tissues.

• Consider post-transcriptional regulation: Investigate potential miRNA targeting, RNA stability, or alternative splicing affecting AUG8 mRNA.

• Examine post-translational modifications: Use phospho-specific or ubiquitin-specific antibodies to detect modified forms of AUG8.

• Protein stability analysis: Perform cycloheximide chase experiments to determine AUG8 protein half-life.

• Subcellular fractionation: Check if protein localization affects detection in different cellular compartments.

The systematic approach should include replicate experiments with both methods, potentially different antibody lots, and validation with alternative techniques such as mass spectrometry .

What statistical approaches are recommended for quantifying AUG8 immunofluorescence signal changes across experimental conditions?

For rigorous quantification of AUG8 immunofluorescence signals:

• Image acquisition standardization:

  • Use identical microscope settings for all samples

  • Collect z-stacks of appropriate depth (0.3-0.5μm intervals)

  • Include fluorescence standards for calibration

• Recommended quantification methods:

  • Integrated density measurements of defined ROIs

  • Background subtraction using adjacent negative regions

  • Signal-to-noise ratio calculations

• Statistical analysis workflow:

  • Normality testing (Shapiro-Wilk test)

  • For normal distributions: ANOVA with post-hoc tests (Tukey's HSD)

  • For non-normal distributions: Kruskal-Wallis with Mann-Whitney U tests

  • Minimum sample size: 30-50 cells from ≥3 biological replicates

• Data visualization:

  • Box plots showing median, quartiles, and outliers

  • Violin plots for distribution patterns

  • Include individual data points for transparency

For colocalization analysis with other proteins, use Pearson's or Mander's correlation coefficients with appropriate thresholding. Report Cohen's d for effect size alongside p-values .

How does AUG8 antibody specificity compare across different plant species beyond Arabidopsis?

AUG8 antibody (CSB-PA154725XA01DOA) cross-reactivity across plant species correlates with evolutionary conservation of the AUG8 protein sequence:

The antibody targets a region in AUG8 that shows higher sequence divergence in monocots compared to dicots. When working with non-Arabidopsis species, researchers should:

• Perform western blots to confirm specific binding at the expected molecular weight

• Include peptide competition assays

• Consider using alternative antibodies raised against conserved epitopes for cross-species studies

• Validate with recombinant proteins from the species of interest

What approaches should researchers take to minimize cross-reactivity with other augmin complex proteins?

To minimize cross-reactivity with other augmin complex proteins:

• Epitope selection strategy: The CSB-PA154725XA01DOA antibody targets unique regions of AUG8 that have minimal sequence homology with other augmin subunits (AUG1-7). Focus on the C-terminal region of AUG8 which shows the highest divergence.

• Pre-absorption protocol: Incubate the antibody with recombinant AUG6 and AUG7 proteins (the most structurally similar augmin subunits) at a 10:1 (protein:antibody) ratio for 2 hours at room temperature before use.

• Differentiating signal confirmation: Use dual-labeling with antibodies against other augmin subunits to distinguish between specific and non-specific signals.

• Validation in genetic backgrounds: Compare antibody recognition patterns in wild-type plants versus aug8 knockouts and plants overexpressing specific augmin subunits.

• Sequential immunoprecipitation: For interaction studies, perform sequential IPs to remove potential cross-reactive complexes before the main experimental IP.

Researchers should always include negative controls using pre-immune serum and perform western blots to confirm antibody specificity by molecular weight .

What methods can researchers use to identify potential off-target binding of AUG8 antibody in complex plant extracts?

To identify potential off-target binding of AUG8 antibody in complex plant extracts:

• Immunoprecipitation followed by mass spectrometry:

  • Perform standard immunoprecipitation with AUG8 antibody

  • Analyze precipitated proteins by LC-MS/MS

  • Compare results from wild-type and aug8 mutant samples

  • Proteins present in both samples may represent off-targets

• Two-dimensional western blotting:

  • Separate proteins by both isoelectric point and molecular weight

  • Perform western blotting with AUG8 antibody

  • Compare with theoretical position of AUG8

  • Additional spots indicate potential cross-reactivity

• Protein array screening:

  • Use recombinant protein arrays containing Arabidopsis proteins

  • Probe with AUG8 antibody to identify all potential binding partners

  • Validate findings with reciprocal co-IP experiments

• Competitive binding assays:

  • Pre-incubate antibody with increasing concentrations of purified AUG8 protein

  • Apply to western blots or immunostaining

  • Signals that remain despite competition represent non-specific binding

For greater confidence, combine multiple approaches and validate findings across different experimental conditions .

How can AUG8 antibody be utilized in studying plant responses to abiotic stress conditions?

For studying AUG8's role in abiotic stress responses:

• Differential localization analysis:

  • Subject plants to various stresses (drought, cold, salt, heat)

  • Compare AUG8 localization patterns before and after stress

  • Quantify changes in nuclear vs. cytoplasmic distribution

  • Co-label with stress-responsive proteins to identify potential interactions

• Phosphorylation-specific detection:

  • Generate phospho-specific AUG8 antibodies targeting key residues

  • Monitor phosphorylation status changes under stress conditions

  • Combine with phosphatase inhibitor treatments to preserve modifications

• Microtubule array reorganization:

  • Use dual immunostaining with AUG8 and tubulin antibodies

  • Analyze colocalization coefficients during stress responses

  • Quantify microtubule density, orientation, and stability in relation to AUG8 localization

• Chromatin association studies:

  • Perform ChIP-seq to identify stress-responsive genes associated with AUG8

  • Compare binding profiles under normal and stress conditions

  • Validate with reporter gene assays

The optimal protocol includes time-course experiments capturing early (0-30 minutes), intermediate (1-6 hours), and late (12-48 hours) responses to identify dynamic changes in AUG8 function during stress adaptation .

What are the methodological considerations when using AUG8 antibody in co-immunoprecipitation studies with potential interacting proteins?

For successful co-immunoprecipitation studies with AUG8 antibody:

• Buffer optimization matrix:

• Antibody immobilization approaches:

  • Direct coupling to activated beads (high efficiency but may affect epitope)

  • Protein A/G-mediated binding (versatile but less stable)

  • Biotinylated antibody with streptavidin beads (strongest interaction)

• Pre-clearing strategy:

  • Pre-clear lysates with isotype-matched control antibody

  • Include 1-2 hour incubation with beads alone before antibody addition

  • Add 1% BSA to reduce non-specific binding

• Elution methods comparison:

  • Peptide competition elution (gentlest, preserves interactions)

  • pH gradient elution (intermediate stringency)

  • SDS elution (strongest, disrupts all interactions)

• Controls framework:

  • Input lysate (5-10%)

  • IgG-matched negative control

  • Reverse co-IP with antibodies against interacting partners

  • aug8 knockout negative control

For studying transient or weak interactions, consider crosslinking with DSP (dithiobis(succinimidyl propionate)) before cell lysis .

How can researchers effectively integrate AUG8 antibody-based data with transcriptomics and proteomics data in systems biology approaches?

For integrating AUG8 antibody data with -omics approaches:

• Multi-modal data integration workflow:

  • Generate AUG8 ChIP-seq, RNA-seq, and proteomics data from identical experimental conditions

  • Normalize datasets using appropriate batch correction methods

  • Apply time-series analysis for dynamic studies

• Network construction methodology:

  • Use AUG8 ChIP-seq peaks to identify direct target genes

  • Correlate with RNA-seq expression changes in aug8 mutants

  • Integrate protein interaction data from co-IP/MS experiments

  • Apply Bayesian network algorithms to infer causal relationships

• Validation strategy:

  • Select key nodes from network analysis for functional validation

  • Generate reporter constructs for AUG8-bound promoters

  • Perform directed protein-protein interaction studies

  • Use CRISPR/Cas9 to mutate specific binding sites

• Visualization and analysis platforms:

  • Cytoscape with EnrichmentMap for network visualization

  • Gene Ontology enrichment analysis for functional clustering

  • GSEA for pathway analysis of AUG8-associated genes

  • STRING database integration for protein interaction networks

• Statistical approaches:

  • Apply false discovery rate correction across all -omics datasets

  • Use ANOVA models to identify significant condition-dependent changes

  • Implement machine learning algorithms to classify AUG8-dependent responses

This integrative approach should include at least three biological replicates per condition and appropriate controls for each experimental method .