ACX3.2 Antibody

What is ACX3.2 Antibody and what specific target does it recognize?

ACX3.2 Antibody is a rabbit polyclonal antibody that specifically recognizes ACX6 (acyl-CoA oxidase 6) in Arabidopsis thaliana . This antibody targets plant antigens and is designed to detect proteins involved in fatty acid β-oxidation pathways. ACX3.2 corresponds to the T2D23.2 gene product, which functions in peroxisomal fatty acid metabolism . The antibody has been validated for several immunological techniques and is purified through antigen affinity methods to ensure specificity .

What are the validated applications for ACX3.2 Antibody?

The ACX3.2 Antibody has been validated for multiple research applications, including:

• Enzyme Immunoassays (EIA)

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blot (WB) analysis

• General immunoassay techniques

In Western blot applications, the antibody has been confirmed to generate positive signals when used against the recombinant immunogen protein/peptide . This versatility makes it a valuable tool for researchers investigating acyl-CoA oxidase expression and function in plant systems.

Why study acyl-CoA oxidases in plant research?

Acyl-CoA oxidases (ACOXs) play crucial roles in plant metabolism as they catalyze the first step in fatty acid β-oxidation. Similar to what has been observed in other organisms like the lipolytic yeast Candida aaseri SH14, which contains three Acyl-CoA oxidases encoded by different genes (CaAOX2, CaAOX4, and CaAOX5) , plants utilize these enzymes for breaking down fatty acids for energy production and metabolite synthesis. Understanding ACX proteins provides insights into:

How is the ACX3.2 Antibody formulated for laboratory use?

The ACX3.2 Antibody is formulated with 50% glycerol and contains 0.03% Proclin 300 as a preservative . This formulation helps maintain antibody stability during storage and prevents microbial contamination. The IgG isotype antibody is provided in a non-conjugated format, making it versatile for different detection systems depending on research needs .

How can ACX3.2 Antibody be used to elucidate plant lipid metabolism pathways?

Researchers can employ ACX3.2 Antibody to investigate the complex network of lipid metabolism in Arabidopsis through multiple approaches:

• Tissue-specific expression analysis: Using immunohistochemistry techniques adapted from those used with other antibodies such as P2X3 receptor antibodies , researchers can localize ACX6 expression across different plant tissues and developmental stages.

• Metabolic flux studies: By combining ACX3.2 Antibody detection with metabolomic approaches, researchers can correlate protein expression levels with changes in fatty acid oxidation rates.

• Protein-protein interaction studies: Immunoprecipitation using ACX3.2 Antibody can help identify binding partners of ACX6, revealing regulatory networks in peroxisomal metabolism.

• Stress response investigations: Western blot analysis using this antibody can quantify changes in ACX6 expression under various environmental stresses, similar to methodology used in other protein expression studies .

What experimental design considerations should be addressed when using ACX3.2 Antibody in knockout/mutation studies?

When designing experiments involving ACX3.2 Antibody in knockout or mutation studies:

• Genetic validation: Confirm the genotype of mutant lines using PCR before antibody studies.

• Protein expression confirmation: Use the ACX3.2 Antibody in Western blot analysis to confirm the absence or alteration of the target protein in mutant lines.

• Functional complementation: Design experiments where the antibody can be used to detect reintroduced wild-type or modified ACX6 protein in mutant backgrounds.

• Control selection: Include appropriate wild-type controls processed simultaneously with mutant samples to ensure comparable detection conditions.

• Cross-reactivity assessment: Test for potential cross-reactivity with related ACX proteins, especially in studies of compensatory expression in ACX6 mutants.

How does ACX3.2 Antibody perform in studies of plant signaling pathways related to lipid metabolism?

ACX3.2 Antibody can be effectively used to investigate signaling pathways through several approaches:

• Phosphorylation studies: By combining ACX3.2 Antibody detection with phospho-specific antibodies, researchers can determine if regulatory phosphorylation affects ACX6 function, similar to approaches used in adipocyte differentiation studies with PPARγ and MAPK pathways .

• Hormone response analysis: Western blot analysis using ACX3.2 Antibody can reveal changes in ACX6 expression following treatment with plant hormones, providing insights into regulatory mechanisms.

• Transcriptional regulation: Comparing protein detection using ACX3.2 Antibody with RT-PCR data can elucidate post-transcriptional regulation mechanisms affecting ACX6 levels.

• Environmental stress response: Quantitative analysis of ACX6 protein levels using ACX3.2 Antibody under different stress conditions can reveal functional adaptations in fatty acid metabolism.

What are the optimal Western blot conditions when using ACX3.2 Antibody?

For optimal Western blot results with ACX3.2 Antibody, follow these guidelines:

• Sample preparation:

  • Extract proteins using a buffer containing protease inhibitors

  • Determine protein concentration using BCA or Bradford assay

  • Load 20-50 μg of total protein per lane

• Gel electrophoresis:

  • Use 10% SDS-PAGE gels for optimal separation

  • Include molecular weight markers

• Transfer conditions:

  • Transfer to PVDF membrane at 100V for 1 hour or 30V overnight

  • Verify transfer with reversible staining

• Blocking:

  • Block with 3-5% non-fat dry milk in TBS-T (similar to protocols used in other Western blot procedures )

  • Alternatively, use 3% BSA in TBS-T for reduced background

• Antibody incubation:

  • Dilute primary ACX3.2 Antibody 1:1000 to 1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash thoroughly with TBS-T (3 × 10 minutes)

  • Incubate with appropriate HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) detection system

  • Optimize exposure time to avoid saturation

  • Consider using digital imaging systems for quantification

How should samples be prepared for ELISA using ACX3.2 Antibody?

For ELISA applications with ACX3.2 Antibody:

• Sample preparation:

  • For plant tissue: Homogenize in phosphate-buffered saline (PBS) with protease inhibitors

  • Centrifuge at 12,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• Plate coating:

  • For direct ELISA: Coat plates with 1-10 μg/ml of sample protein in coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6)

  • For sandwich ELISA: Coat with a capture antibody against ACX6 or a related protein

• Blocking:

  • Block with 3% BSA in PBS for 1-2 hours at room temperature

• Antibody application:

  • For direct ELISA: Apply ACX3.2 Antibody at 1:1000 dilution

  • For sandwich ELISA: Apply sample first, then ACX3.2 Antibody

• Detection:

  • Use appropriate HRP-conjugated secondary antibody

  • Develop with TMB substrate and measure absorbance at 450 nm

• Controls:

  • Include positive control (purified ACX6 protein if available)

  • Include negative control (samples from ACX6 knockout plants)

  • Include blank wells for background subtraction

What approaches can be used to validate the specificity of ACX3.2 Antibody?

Validating antibody specificity is crucial for reliable research results. For ACX3.2 Antibody, consider these approaches:

• Genetic validation:

  • Compare Western blot results between wild-type and ACX6 knockout plants

  • Expected result: Signal should be absent or significantly reduced in knockout samples

• Peptide competition assay:

  • Pre-incubate ACX3.2 Antibody with excess immunizing peptide

  • Apply to Western blot alongside untreated antibody

  • Expected result: Specific signal should be blocked in the pre-incubated sample, similar to techniques used with P2X3 receptor antibodies

• Recombinant protein validation:

  • Test antibody against purified recombinant ACX6 protein

  • Expected result: Single band at the expected molecular weight

• Mass spectrometry validation:

  • Perform immunoprecipitation using ACX3.2 Antibody

  • Analyze precipitated proteins by mass spectrometry

  • Expected result: ACX6 protein should be identified as the predominant protein

• Cross-reactivity testing:

  • Test against other ACX family members (if available)

  • Expected result: Minimal or no detection of other family members

How can researchers address weak or absent signals when using ACX3.2 Antibody?

When encountering weak or absent signals with ACX3.2 Antibody, consider these troubleshooting approaches:

• Protein extraction optimization:

  • Ensure complete protein extraction with appropriate buffers

  • Add protease inhibitors to prevent degradation

  • Consider different extraction methods for membrane-associated proteins

• Antibody concentration adjustment:

  • Increase primary antibody concentration (try 1:500 or 1:200)

  • Increase incubation time to overnight at 4°C

  • Consider using signal enhancement systems

• Detection system sensitivity:

  • Switch to more sensitive detection reagents

  • Increase substrate incubation time

  • Consider different membrane types (PVDF vs. nitrocellulose)

• Sample handling improvements:

  • Avoid freeze-thaw cycles of samples

  • Process samples immediately after collection

  • Keep samples cold throughout preparation

• Protein denaturation considerations:

  • Test different denaturation conditions (boiling time, reducing agents)

  • For membrane proteins, avoid excessive boiling that may cause aggregation

How can researchers resolve conflicting data when using ACX3.2 Antibody across different experimental platforms?

When facing contradictory results across different experimental platforms:

• Platform-specific optimization:

  • Adjust antibody concentrations for each platform independently

  • Optimize detection methods for each specific technique

  • Validate antibody performance on each platform separately

• Sample preparation consistency:

  • Standardize protein extraction methods across experiments

  • Use the same buffer systems when possible

  • Process all comparative samples simultaneously

• Control implementation:

  • Include consistent positive and negative controls across all platforms

  • Use recombinant protein standards when available

  • Include internal loading controls appropriate for each platform

• Quantification methods:

  • Apply appropriate normalization techniques for each platform

  • Use multiple quantification methods to verify results

  • Apply statistical analyses to determine significance of differences

• Biological validation:

  • Confirm key findings using complementary approaches

  • Consider genetic approaches (RNAi, CRISPR) to validate antibody results

  • Compare protein and mRNA levels to identify post-transcriptional regulation

What statistical approaches are appropriate for analyzing ACX3.2 Antibody signal quantification?

For rigorous quantitative analysis of ACX3.2 Antibody signals:

• Normalization approaches:

  • Normalize to housekeeping proteins (e.g., actin, similar to methods used in adipocyte differentiation studies )

  • Consider total protein normalization using stain-free technology

  • Use multiple reference proteins for more reliable normalization

• Replicate design:

  • Perform at least three biological replicates

  • Include multiple technical replicates within each biological replicate

  • Apply appropriate statistical tests based on experimental design

• Statistical analysis methods:

  • For comparing two conditions: t-test (paired or unpaired as appropriate)

  • For multiple conditions: ANOVA with appropriate post-hoc tests

  • For non-normally distributed data: Non-parametric alternatives

• Regression analysis:

  • For dose-response studies: Apply appropriate regression models

  • For time-course experiments: Consider repeated measures analysis

• Data reporting:

  • Report means with standard deviation or standard error

  • Include p-values and significance thresholds

  • Present normalized data alongside representative raw data images

How does ACX3.2 Antibody compare to other antibodies targeting acyl-CoA oxidases?

When considering ACX3.2 Antibody in relation to other ACX-targeting antibodies:

• Target specificity comparison:

  • ACX3.2 Antibody targets specifically the ACX6 isoform in Arabidopsis thaliana

  • Other antibodies may target different ACX family members or be cross-reactive

  • Some commercial antibodies target conserved regions present in multiple ACX proteins

• Species reactivity:

  • ACX3.2 Antibody is validated for Arabidopsis thaliana

  • Compare with antibodies validated for other plant species or model organisms

  • Consider evolutionary conservation when evaluating cross-species applications

• Performance characteristics:

  • Compare sensitivity limits across different antibodies

  • Evaluate specificity using knockout controls

  • Compare signal-to-noise ratios in common applications

• Application versatility:

  • ACX3.2 Antibody is validated for ELISA and Western blot

  • Some alternatives may offer additional validated applications

  • Consider needs for specialized applications like immunohistochemistry or flow cytometry

What complementary research tools work effectively with ACX3.2 Antibody for comprehensive metabolic studies?

For comprehensive studies of plant metabolism using ACX3.2 Antibody:

• Complementary antibodies:

  • Antibodies against other β-oxidation enzymes

  • Antibodies against peroxisomal marker proteins

  • Antibodies against regulatory proteins in lipid metabolism

• Metabolic analysis tools:

  • Metabolomic profiling platforms to measure fatty acid intermediates

  • Isotope labeling approaches to track metabolic flux

  • Enzyme activity assays to correlate protein levels with function

• Transcriptomic approaches:

  • RT-qPCR for targeted gene expression analysis

  • RNA-seq for global transcriptional responses

  • Compare protein levels (via ACX3.2 Antibody) with mRNA expression

• Genetic resources:

  • ACX6 mutant lines for functional validation

  • Overexpression lines to study gain-of-function phenotypes

  • Reporter constructs for localization and expression studies

• Imaging technologies:

  • Confocal microscopy for subcellular localization studies

  • In vivo imaging systems for temporal expression patterns

  • Super-resolution approaches for detailed structural analysis

How can researchers integrate ACX3.2 Antibody data with systems biology approaches?

Integrating ACX3.2 Antibody data into systems biology frameworks:

• Multi-omics integration:

  • Correlate protein levels detected by ACX3.2 Antibody with transcriptomic data

  • Integrate with metabolomic profiles of fatty acids and related metabolites

  • Combine with proteomic data to identify co-regulated protein networks

• Pathway modeling:

  • Incorporate ACX6 protein levels into models of fatty acid β-oxidation

  • Develop kinetic models incorporating enzyme abundance data

  • Perform flux balance analysis with constraints derived from antibody quantification

• Network analysis:

  • Identify hub proteins and regulatory nodes connected to ACX6

  • Perform gene ontology enrichment analysis of co-expressed proteins

  • Construct protein-protein interaction networks centered on ACX6

• Temporal and spatial mapping:

  • Map ACX6 expression across developmental stages using ACX3.2 Antibody

  • Correlate with tissue-specific metabolic profiles

  • Develop spatiotemporal models of fatty acid metabolism

• Comparative systems analysis:

  • Compare ACX6 regulation across multiple plant species

  • Analyze evolutionary conservation of regulatory mechanisms

  • Identify species-specific adaptations in fatty acid metabolism