AXS2 Antibody

What is AXS2 and what organisms express this protein?

AXS2 (UDP-D-apiose) is a plant protein primarily studied in Arabidopsis thaliana (Mouse-ear cress). The commercially available AXS2 antibody is typically raised in rabbits using recombinant Arabidopsis thaliana AXS2 protein as the immunogen . This polyclonal antibody is designed for research applications in plant biology and is not intended for diagnostic or therapeutic procedures .

What are the standard applications for AXS2 antibody in research?

AXS2 antibody is primarily validated for ELISA and Western blotting (WB) applications . The antibody is supplied in liquid form with a storage buffer containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) . Most commercial preparations are antigen-affinity purified to ensure specificity in these applications.

How does antibody validation differ between plant and human protein targets?

While plant-specific antibodies like AXS2 and human-targeted antibodies follow similar validation principles, plant antibodies often require additional controls due to cell wall components that can interfere with antibody-antigen interactions. Validation typically involves confirming specificity through knockout lines or competing antigen tests. Human antibodies, like those targeting ASXL2, often undergo comprehensive validation across multiple applications including IHC, ICC-IF, and WB to ensure reproducibility .

How can researchers optimize AXS2 antibody use in Western blotting applications?

For optimal Western blotting results with AXS2 antibody:

Researchers should include appropriate positive controls (Arabidopsis thaliana extract) and negative controls (non-plant extracts) to validate specificity .

What experimental controls are essential when designing studies using AXS2 antibody?

When designing experiments with AXS2 antibody, the following controls are essential:

• Primary Antibody Controls: Include samples processed without primary antibody to assess secondary antibody non-specific binding

• Competitive Inhibition: Pre-incubate AXS2 antibody with excess antigen to confirm signal specificity

• Isotype Controls: Use non-specific rabbit IgG at equivalent concentrations to assess background

• Cross-Reactivity Assessment: Test antibody against similar plant species to determine specificity beyond Arabidopsis

• Knockout/Knockdown Controls: Where available, include AXS2-deficient samples to confirm antibody specificity

How do researchers address potential cross-reactivity in antibody-based detection systems?

Cross-reactivity remains a significant challenge in antibody-based research. For plant antibodies like AXS2, researchers should:

• Perform sequence alignment of the target protein across related species to predict potential cross-reactivity

• Execute Western blot analysis using extracts from multiple plant species to empirically determine cross-reactivity

• Employ epitope mapping to identify the specific binding region of the antibody

• Consider absorption studies where the antibody is pre-incubated with proteins from non-target species

• Implement immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody

What storage and handling practices maximize AXS2 antibody stability and performance?

To maintain optimal antibody activity:

• Upon receipt, aliquot the antibody to minimize freeze-thaw cycles and store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles which significantly reduce antibody activity

• For working solutions, store at 4°C for up to one week

• If precipitation occurs, centrifuge the antibody solution before use

• Document lot numbers and maintain validation data for reproducibility

• Consider adding carrier proteins (BSA) to diluted antibody solutions to improve stability

How can researchers troubleshoot non-specific binding when using AXS2 antibody?

Non-specific binding is a common challenge in antibody-based techniques. Effective troubleshooting approaches include:

• Increase blocking time and concentration: Use 5-10% blocking agent for 1-2 hours at room temperature

• Optimize antibody dilution: Test serial dilutions to identify optimal concentration

• Add detergent: Increase Tween-20 concentration (up to 0.1%) in wash buffers to reduce hydrophobic interactions

• Change blocking agent: Switch between BSA, non-fat milk, or commercial blocking reagents

• Reduce incubation temperature: Perform antibody incubation at 4°C to increase binding specificity

• Pre-absorb antibody: Incubate with non-target tissue extract to remove cross-reactive antibodies

What approaches help researchers validate antibody specificity in complex biological samples?

Validating antibody specificity requires multiple complementary approaches:

• Multi-technique confirmation: Verify target detection across different methods (WB, ELISA, IHC)

• Size validation: Confirm detected protein matches expected molecular weight

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific signals

• Genetic models: Test antibody in knockout/knockdown systems

• Orthogonal targeting: Use antibodies targeting different epitopes of the same protein

• Signal correlation: Compare antibody signal with mRNA expression patterns

How do antibody-based detection systems differ between plant and human research contexts?

Plant and human antibody research share fundamental principles but differ in several key aspects:

Human antibody research, such as with ASXL2 antibodies, benefits from more extensive validation resources and standardized protocols compared to plant antibody research .

What role do antibodies play in studying protein-protein interactions in research models?

Antibodies are critical tools for studying protein-protein interactions in both plant and human systems. They enable:

• Co-immunoprecipitation (Co-IP): Capturing protein complexes for further analysis

• Proximity Ligation Assays (PLA): Visualizing interactions in situ with spatial resolution

• Chromatin Immunoprecipitation (ChIP): Studying protein-DNA interactions

• Pull-down Assays: Identifying binding partners of target proteins

• FRET/BRET Analysis: When coupled with fluorescent tags, enabling dynamic interaction studies

This methodology has been demonstrated in studies of protein-protein interactions like the SARS-CoV-2 Spike protein with ACE2 receptor, where antibodies were crucial for binding assays and structural analyses .

How do researchers integrate antibody-based findings with other molecular techniques?

Modern research requires integrating antibody-based techniques with complementary approaches:

• Transcriptomic Correlation: Compare protein detection with RNA-seq data to validate findings

• Mass Spectrometry Validation: Confirm antibody-detected proteins through peptide identification

• Functional Assays: Couple antibody detection with activity assays to correlate presence and function

• Imaging Techniques: Combine immunofluorescence with advanced microscopy for spatial context

• Genetic Manipulation: Correlate antibody detection with phenotypic changes following gene editing

How are new antibody validation standards improving research reproducibility?

Emerging standards for antibody validation are enhancing reproducibility through:

• Application-specific Validation: Requiring evidence for each specific use case

• Genetic Strategy: Testing in knockout/knockdown models

• Independent Antibody Strategy: Comparing results from antibodies targeting different epitopes

• Orthogonal Strategy: Correlating protein and mRNA levels

• Expression of Tagged Proteins: Using tags to validate antibody detection

These approaches address historical issues with antibody reliability and are being adopted by leading antibody providers to ensure consistent experimental outcomes .

What emerging technologies are enhancing antibody-based detection systems?

Cutting-edge technologies improving antibody research include:

• Single-cell Western Blotting: Enabling protein analysis at single-cell resolution

• Multiplexed Immunofluorescence: Detecting multiple targets simultaneously

• Antibody Engineering: Creating recombinant antibodies with improved specificity

• Microfluidic Immunoassays: Reducing sample volume requirements while increasing throughput

• AI-assisted Epitope Design: Computational approaches to improved antibody development

These advances are creating new possibilities for detecting low-abundance proteins and analyzing heterogeneous samples with unprecedented precision.