The Augustine (AUG) blood group system involves four high- or low-frequency antigens (AUG1–AUG4) located on the ENT1 protein encoded by SLC29A1. These antigens are clinically significant in transfusion medicine and pregnancy:
AUG2 (At<sup>a</sup>) antibodies have caused hemolytic transfusion reactions .
AUG3 antibodies were linked to severe hemolytic disease of the fetus and newborn .
No AUG8 antigen or antibody is documented in this system. The numbering (AUG1–AUG4) reflects the order of discovery, not a sequence extending to "AUG8" .
Anti-AAV8 neutralizing antibodies (NAbs) target adeno-associated virus serotype 8 (AAV8), a vector used in gene therapy. Key findings from a multicenter study of hemophilia patients include :
Parameter | AAV8 NAbs Prevalence (%) | AAV2 NAbs Prevalence (%) | AAV5 NAbs Prevalence (%) |
---|---|---|---|
Baseline | 46.9 | 53.1 | 53.4 |
Year 1 | 47.2 | 50.0 | 52.8 |
Year 2 | 46.7 | 51.1 | 51.1 |
These antibodies persist over time and impact eligibility for AAV-based gene therapies .
This monoclonal antibody targets 8-oxoguanine (8-oxo-G), a DNA lesion caused by oxidative stress. Key characteristics :
Property | Detail |
---|---|
Host Species | Mouse |
Clonality | Monoclonal (Clone 2Q2311) |
Isotype | IgM |
Applications | ELISA, IHC, ICC/IF |
Reactivities | Modified amino acids (human samples) |
Citations | 12 publications, 1 independent review |
It is used to detect oxidative DNA damage in research contexts, such as in HeLa cells .
Terminology Confusion: "AUG8" may result from conflating:
Database Coverage: The Antibody Registry catalogs over 2.5 million antibodies but lists no entries for "AUG8" .
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 .
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 .
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 .
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 .
Optimal parameters for AUG8 immunohistochemistry vary by tissue type:
Tissue Type | Fixation | Antigen Retrieval | Antibody Dilution | Incubation Conditions | Detection System |
---|---|---|---|---|---|
Root tips | 4% PFA, 2h | 10mM citrate buffer, pH 6.0, 95°C, 10min | 1:200 | 16h at 4°C | Fluorescent secondary 1:500 |
Leaf tissue | 4% PFA, 4h | 10mM citrate buffer, pH 6.0, 95°C, 15min | 1:100 | 24h at 4°C | Fluorescent secondary 1:400 |
Meristematic tissue | 4% PFA, 2h | 10mM Tris-EDTA, pH 9.0, 95°C, 15min | 1:150 | 18h at 4°C | Fluorescent secondary 1:500 |
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 .
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 .
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
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 .
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 .
AUG8 antibody (CSB-PA154725XA01DOA) cross-reactivity across plant species correlates with evolutionary conservation of the AUG8 protein sequence:
Plant Species | Cross-Reactivity | Required Antibody Dilution | Notes |
---|---|---|---|
Arabidopsis thaliana | High (primary target) | 1:200-1:500 | Optimal specificity |
Brassica species | Moderate-High | 1:100-1:200 | Recommended for close relatives |
Rice (Oryza sativa) | Low-Moderate | 1:50-1:100 | May require validation |
Tomato (Solanum lycopersicum) | Very Low | 1:25-1:50 | Not recommended |
Wheat (Triticum aestivum) | Minimal | Not recommended | Use species-specific antibodies |
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
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 .
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 .
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 .
For successful co-immunoprecipitation studies with AUG8 antibody:
Buffer optimization matrix:
Buffer Component | Recommended Range | Stringent Conditions | Mild Conditions |
---|---|---|---|
Salt (NaCl) | 100-300mM | 300mM | 100mM |
Detergent | 0.1-1% NP-40 or Triton X-100 | 1% | 0.1% |
pH | 7.0-8.0 | 7.0 | 7.5-8.0 |
Divalent cations | 1-5mM MgCl₂ | None | 2-5mM |
Reducing agents | 0.5-5mM DTT | 0.5mM | 5mM |
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 .
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 .