AUG6 Antibody

What is AUG6 Antibody and what is its target protein?

AUG6 Antibody (CSB-PA297044XA01DOA) is a rabbit-derived polyclonal antibody that specifically targets the AUG6 protein (UniProt: Q94BP7) from Arabidopsis thaliana . This antibody is designed for research applications in plant molecular biology, particularly in studies examining microtubule-organizing centers, as AUG6 is part of the augmin complex involved in microtubule nucleation. The antibody is raised against a recombinant Arabidopsis thaliana AUG6 protein as its immunogen, making it particularly valuable for studying this specific plant protein .

What experimental applications is AUG6 Antibody validated for?

Based on vendor specifications, AUG6 Antibody has been tested and validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) applications . The antibody's performance in these applications suggests its suitability for detecting AUG6 protein in complex protein mixtures from Arabidopsis samples. When selecting an antibody for specific experimental applications, researchers should determine whether the antibody has been characterized for their intended use, as antibody performance can vary significantly across different experimental conditions and protocols .

What are the optimal storage conditions for AUG6 Antibody?

Upon receipt, AUG6 Antibody should be stored at either -20°C or -80°C to maintain its efficacy and specificity . Importantly, researchers should avoid repeated freeze-thaw cycles, as these can significantly degrade antibody quality and performance. For long-term storage, aliquoting the antibody into single-use volumes is recommended to minimize freeze-thaw damage. The antibody is provided in liquid form with an appropriate storage buffer, which helps maintain stability during proper storage .

What strategies should I use to validate AUG6 Antibody specificity?

To thoroughly validate AUG6 Antibody specificity, implement multiple complementary approaches from the "five pillars" of antibody validation:

For maximum confidence, implement at least two validation strategies. For instance, combining genetic strategy (using aug6 mutant plants as negative controls) with an orthogonal approach (correlating protein detection with transcriptomic data) provides robust validation of specificity .

How can I determine if AUG6 Antibody is suitable for my specific experimental conditions?

Antibody suitability for specific experimental conditions requires contextual validation. For AUG6 Antibody:

• Perform preliminary tests under your exact experimental conditions, including buffer compositions, incubation times, and temperatures.

• Include positive controls (samples known to express AUG6) and negative controls (samples lacking AUG6 expression) specific to your experimental system.

• Titrate antibody concentrations to determine optimal signal-to-noise ratios.

• Validate protocol-specific parameters (e.g., fixation methods for immunofluorescence or blocking conditions for Western blotting).

• Document all optimization steps methodically to ensure reproducibility .

Remember that antibody performance is context-dependent, and characterization data from vendors may not precisely translate to your specific experimental setup. End-user validation is essential for each specific use and experimental condition .

What are the optimal Western blotting conditions for AUG6 Antibody?

While exact optimal conditions may require empirical determination, the following protocol provides a starting point for Western blotting with AUG6 Antibody:

• Sample preparation: Extract proteins from Arabidopsis tissues using a plant-specific lysis buffer containing protease inhibitors to prevent degradation of AUG6 protein.

• Protein separation: Load 20-50 μg of total protein per lane on a 10-12% SDS-PAGE gel.

• Transfer: Use PVDF membrane for optimal protein binding during wet transfer (100V for 60-90 minutes).

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

• Primary antibody: Dilute AUG6 Antibody 1:500 to 1:2000 in blocking buffer; incubate overnight at 4°C.

• Washing: Wash membrane with TBST (3 × 10 minutes).

• Secondary antibody: Use anti-rabbit HRP-conjugated antibody at 1:5000 to 1:10000; incubate for 1 hour at room temperature.

• Detection: Develop using ECL substrate, optimizing exposure time to avoid signal saturation.

Include both positive controls (wild-type Arabidopsis samples) and negative controls (aug6 mutant samples when available) to verify specificity .

How should I optimize AUG6 Antibody for immunofluorescence applications?

Although AUG6 Antibody is not explicitly validated for immunofluorescence by the manufacturer , researchers may optimize this application through:

• Fixation method testing: Compare paraformaldehyde (4%), methanol, and acetone fixation to determine which best preserves AUG6 epitopes.

• Antigen retrieval: Evaluate whether heat-induced or enzymatic antigen retrieval improves signal detection.

• Permeabilization: Optimize cell wall and membrane permeabilization using 0.1-0.5% Triton X-100 or plant-specific cell wall degrading enzymes.

• Antibody concentration: Test a range of primary antibody dilutions (1:100 to 1:1000) and incubation times (overnight at 4°C is often optimal).

• Validation controls: Include:

  • Peptide competition assays

  • aug6 mutant samples as negative controls

  • Dual-labeling with known interacting partners

  • Secondary antibody-only controls to assess background

Document all optimization parameters systematically to establish a reproducible protocol.

How can I troubleshoot weak or absent signals when using AUG6 Antibody?

When facing challenges with signal detection using AUG6 Antibody, systematically evaluate:

• Antibody integrity: Verify storage conditions; consider obtaining a new antibody lot if degradation is suspected.

• Protein abundance: AUG6 may be expressed at low levels; increase protein loading or concentrate samples.

• Epitope accessibility: Test different sample preparation methods:

  • For Western blotting: Adjust reducing conditions or denaturation procedures

  • For immunohistochemistry: Optimize fixation and antigen retrieval

• Detection method sensitivity: Switch to more sensitive detection methods (e.g., from colorimetric to chemiluminescent or fluorescent detection).

• Buffer optimization: Adjust salt concentration, pH, or detergent levels in washing and incubation buffers.

• Incubation parameters: Extend primary antibody incubation time (up to overnight at 4°C) or optimize temperature.

Document each troubleshooting step methodically to identify critical parameters affecting antibody performance .

How should I interpret contradictory results between AUG6 protein detection and transcript analysis?

Discrepancies between protein detection using AUG6 Antibody and transcript levels may result from several biological or technical factors:

• Post-transcriptional regulation: Evaluate potential mechanisms including:

  • microRNA-mediated repression

  • RNA binding protein regulation

  • Altered mRNA stability

• Post-translational modifications: Consider if modifications might affect:

  • Epitope recognition by the antibody

  • Protein stability or half-life

  • Protein localization or compartmentalization

• Technical considerations:

  • Antibody specificity: Validate using knockout/knockdown controls

  • Extraction efficiency: Different protocols may extract proteins with varying efficacy

  • Detection sensitivity thresholds: Protein detection may require more sensitive methods than transcript analysis

To resolve contradictions, implement orthogonal validation strategies that measure AUG6 at different biological levels (transcriptomic, proteomic, and functional) .

What quantification approaches are appropriate for AUG6 Antibody-based protein detection?

For rigorous quantification of AUG6 protein levels:

• Western blot densitometry:

  • Use housekeeping proteins (actin, tubulin) for normalization

  • Ensure signal falls within linear detection range

  • Employ at least three biological replicates

  • Consider using fluorescent secondary antibodies for wider linear range

• ELISA quantification:

  • Develop a standard curve using recombinant AUG6 protein

  • Validate sample matrix effects

  • Include spike recovery controls to assess accuracy

• Flow cytometry (if optimized for plant cells):

  • Use median fluorescence intensity for quantification

  • Include calibration beads to standardize across experiments

  • Perform antibody titration to determine optimal concentration

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design

  • Report both biological and technical variability

  • Consider power analysis to determine appropriate sample sizes

Regardless of method, include proper controls and ensure that quantification remains within the validated linear range of detection .

How should I design experiments to compare AUG6 expression across different developmental stages or treatments?

When designing experiments to examine AUG6 expression patterns:

• Experimental controls:

  • Include wild-type samples alongside treated/developmental samples

  • Consider using aug6 mutant lines (when available) as negative controls

  • Implement appropriate vehicle controls for treatments

• Sampling strategy:

  • Define precise developmental stages using standardized metrics

  • Collect samples at consistent times to account for potential circadian regulation

  • Maintain uniformity in tissue collection, processing, and storage

• Technical considerations:

  • Process all samples simultaneously to minimize batch effects

  • Randomize sample processing order

  • Include inter-experimental calibrators for cross-experiment normalization

• Documentation requirements:

  • Record detailed growth conditions (light, temperature, media composition)

  • Document exact sample collection protocols

  • Maintain comprehensive records of antibody lot numbers and dilutions

• Analysis approach:

  • Normalize AUG6 expression to appropriate reference proteins

  • Apply statistical methods appropriate for the experimental design

  • Consider multiple hypothesis testing correction when examining numerous conditions

This structured approach enhances reproducibility and facilitates meaningful interpretation of AUG6 expression patterns .

What considerations are important when using AUG6 Antibody for co-immunoprecipitation studies?

While AUG6 Antibody has not been specifically validated for immunoprecipitation , researchers interested in studying AUG6 protein interactions should consider:

• Buffer optimization:

  • Test multiple lysis buffers varying in salt concentration and detergent type

  • Evaluate non-denaturing conditions to preserve protein-protein interactions

  • Include protease and phosphatase inhibitors to maintain interaction integrity

• Control experiments:

  • Perform parallel IPs with non-specific IgG from the same species (rabbit)

  • Include aug6 mutant samples as negative controls

  • Consider expressing tagged versions of AUG6 for validation

• Antibody coupling:

  • Determine optimal antibody-to-bead ratio

  • Compare direct coupling versus indirect capture using Protein A/G

  • Evaluate crosslinking to prevent antibody contamination in eluates

• Validation approaches:

  • Confirm AUG6 precipitation by Western blotting a small fraction of the IP

  • Consider mass spectrometry analysis to comprehensively identify interacting partners

  • Validate key interactions through reciprocal co-IP or orthogonal methods

• Data analysis:

  • Apply appropriate statistical threshold for identifying significant interactions

  • Filter results against common contaminant databases

  • Validate novel interactions through independent experimental approaches

These considerations help ensure specific and biologically relevant results when investigating AUG6 protein complexes .

How does antibody validation for AUG6 compare to validation strategies for other research antibodies?

The validation principles for AUG6 Antibody align with broader antibody validation frameworks, though with plant-specific considerations:

• Comparative framework assessment:

  • The "five pillars" validation framework applies universally to AUG6 and other research antibodies, though implementation differs for plant proteins

  • Genetic validation using knockout/knockdown lines is particularly valuable for both plant and mammalian systems

  • Orthogonal validation comparing protein and transcript levels follows similar principles across systems

• Plant-specific challenges:

  • Cell wall barriers require specialized extraction protocols

  • Plant-specific post-translational modifications may affect epitope recognition

  • Specialized fixation protocols for plant tissues may be necessary

  • Lower protein yields often necessitate optimization of detection sensitivity

• Cross-experimental validation:

  • Like other research antibodies, AUG6 Antibody requires validation in each specific experimental context

  • Application-specific controls should be implemented regardless of antibody target

  • Independent replication remains the gold standard for all antibody applications

The fundamental principles of specificity verification, context-dependent validation, and appropriate controls are universally applicable across research antibodies .

What emerging technologies might enhance AUG6 detection and quantification beyond traditional antibody-based methods?

Innovative approaches for studying AUG6 protein include:

• CRISPR-based tagging:

  • Endogenous tagging of AUG6 with fluorescent proteins or epitope tags

  • Avoids potential artifacts from antibody cross-reactivity

  • Enables live-cell imaging of AUG6 dynamics

• Proximity labeling technologies:

  • BioID or TurboID fusion to AUG6 to identify proximal proteins

  • APEX2 fusions for ultrastructural localization studies

  • Spatial and temporal mapping of AUG6 protein communities

• Single-molecule detection methods:

  • Super-resolution microscopy of tagged AUG6 variants

  • Single-molecule pull-down (SiMPull) for quantitative interaction studies

  • Correlative light and electron microscopy for contextual localization

• Quantitative proteomics:

  • Selective reaction monitoring (SRM) for absolute quantification

  • Tandem mass tag (TMT) labeling for multiplexed comparative studies

  • Data-independent acquisition (DIA) for comprehensive protein network analysis

• Nanobody development:

  • Plant-specific nanobodies against AUG6 for improved tissue penetration

  • Intrabody expression for live-cell studies of AUG6 function

  • Multiplexed nanobody panels for simultaneous detection of interaction partners

These emerging approaches complement traditional antibody-based methods and may provide enhanced specificity, sensitivity, and contextual information about AUG6 biology.