DTX25 Antibody

What is DTX25 and what role does it play in Arabidopsis thaliana?

DTX25 (DETOXIFICATION 25) is a protein expressed in Arabidopsis thaliana (Mouse-ear cress), a widely used model organism in plant biology. DTX25 belongs to the MATE (Multidrug And Toxic compound Extrusion) family of transporters that play crucial roles in detoxification processes and secondary metabolite transport in plants. The protein has a UniProt identifier of Q8L616, which can be used to access detailed information about its sequence and predicted functional domains . Understanding DTX25's function is essential for researchers investigating plant stress responses, defense mechanisms, and metabolite transport across cellular membranes. When designing experiments with DTX25 antibody, consider the protein's subcellular localization and tissue-specific expression patterns to properly interpret your results.

What are the recommended storage conditions for DTX25 antibody?

DTX25 antibody should be stored at -20°C or -80°C upon receipt to maintain its activity and specificity. Repeated freeze-thaw cycles should be avoided as they can degrade antibody performance . The antibody is typically supplied in a liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . For extended storage stability:

Always return the antibody to appropriate storage conditions promptly after use, and consider preparing small working aliquots to avoid repeated freeze-thaw cycles that could compromise antibody performance in sensitive applications.

What applications is DTX25 antibody validated for?

The polyclonal DTX25 antibody (CSB-PA131886XA01DOA) has been validated for ELISA and Western blot (WB) applications in Arabidopsis thaliana samples . While these are the primarily validated applications, researchers may need to optimize conditions for their specific experimental systems. When adapting this antibody to other techniques:

Similar to other research antibodies, validation in your specific experimental context is crucial before proceeding with large-scale studies, especially when applying techniques beyond the manufacturer's validated applications .

How can I optimize DTX25 antibody use in Western blot experiments?

Optimizing DTX25 antibody for Western blot requires systematic adjustment of multiple parameters to achieve specific signal detection while minimizing background. Consider the following optimization strategy:

For plant samples, additional optimization may be necessary due to the complex nature of plant extracts. Consider including additional sample preparation steps to remove compounds that may interfere with antibody binding . Progressive optimization through systematic testing of these parameters will help establish robust protocols specific to your experimental system.

What controls should be included when using DTX25 antibody in research?

Including appropriate controls is critical for interpreting DTX25 antibody results correctly. A comprehensive control strategy should include:

• Positive Control: Extract from wild-type Arabidopsis thaliana tissues known to express DTX25

• Negative Control: Extract from DTX25 knockout/knockdown plants (if available)

• Loading Control: Detection of a housekeeping protein (e.g., actin or tubulin) to normalize expression

• Primary Antibody Omission: To assess secondary antibody non-specific binding

• Blocking Peptide Control: Pre-incubation of antibody with immunizing peptide to confirm specificity

A well-designed control panel allows you to confirm antibody specificity and properly interpret experimental results, especially when examining DTX25 expression across different treatment conditions or genetic backgrounds . In systems where genetic modification is challenging, using related plant species with known sequence differences could provide valuable control information.

How do I troubleshoot cross-reactivity issues with DTX25 antibody?

Cross-reactivity can significantly complicate the interpretation of results when working with polyclonal antibodies like anti-DTX25. To troubleshoot and minimize cross-reactivity:

Experimental approaches to identify cross-reactivity include comparing results across different plant species, analyzing reactivity with recombinant proteins of related family members, and using competitive binding assays with the immunizing peptide . When working with samples from species other than Arabidopsis thaliana, sequence alignment of DTX25 homologs can help predict potential cross-reactivity issues.

What considerations are important when using DTX25 antibody in different plant species?

When extending DTX25 antibody use to non-Arabidopsis species, several important considerations should guide experimental design:

• Sequence Homology Analysis: Compare DTX25 protein sequences between Arabidopsis and target species to predict antibody cross-reactivity

• Epitope Conservation: Focus on regions where the antibody was raised (check with manufacturer for epitope information)

• Validation Requirements: More extensive validation is required when working with non-validated species

• Protocol Modifications: Different species may require adjusted extraction buffers, antibody concentrations, and incubation times

• Control Selection: Include both Arabidopsis and target species samples in parallel experiments

What is the recommended protocol for using DTX25 antibody in ELISA?

For effective use of DTX25 antibody in ELISA applications, the following protocol serves as a starting point that can be optimized for specific experimental needs:

Direct ELISA Protocol:

• Coating: Adsorb purified antigen or total protein extract (10-100 μg/ml) in carbonate/bicarbonate buffer (pH 9.6) to microplate wells overnight at 4°C

• Blocking: Block with 5% BSA or 5% non-fat milk in PBS-T for 1-2 hours at room temperature

• Primary Antibody: Apply DTX25 antibody at optimized dilution in blocking buffer for 1-2 hours at room temperature

• Washing: Wash 4-5 times with PBS-T

• Secondary Antibody: Apply appropriate enzyme-conjugated secondary antibody for 1 hour at room temperature

• Development: Add substrate and measure signal according to secondary antibody conjugate specifications

For quantitative analysis, include a standard curve using purified recombinant DTX25 protein if available. Preliminary titration experiments with different antibody dilutions (e.g., 1:500, 1:1000, 1:2000) will help determine optimal working concentrations for specific experimental conditions .

How can I validate DTX25 antibody specificity for my experimental model?

Validating antibody specificity is critical before conducting extensive experiments. For DTX25 antibody, implement a multi-step validation approach:

• Western Blot Analysis: Confirm band size matches predicted molecular weight of DTX25 in your species

• Immunodepletion: Pre-incubate antibody with purified antigen before application to demonstrate signal reduction

• Genetic Validation: Compare wild-type to DTX25 knockout/knockdown plants to confirm signal reduction or loss

• Mass Spectrometry Correlation: Immunoprecipitate with DTX25 antibody and confirm protein identity by mass spectrometry

• Correlation with RNA Expression: Compare protein levels detected by antibody with mRNA levels from RT-PCR or RNA-seq

This comprehensive validation approach provides multiple lines of evidence for antibody specificity that strengthens the reliability of subsequent experimental results . Document validation results thoroughly for publication and protocol development purposes.

How do I quantify DTX25 expression levels using antibody-based methods?

Accurate quantification of DTX25 protein expression requires appropriate experimental design and analysis methods:

For Western blot quantification, use digital image analysis software to measure band intensity, normalizing to housekeeping protein controls. For ELISA, establish standard curves using purified protein when available. In all cases, include multiple biological and technical replicates to ensure statistical robustness . Document all image acquisition parameters and analysis settings to ensure reproducibility.

What are the common challenges in interpreting DTX25 antibody-based experimental results?

Interpreting DTX25 antibody experimental results requires awareness of several potential pitfalls:

• Background vs. Specific Signal: Distinguishing true DTX25 signal from background, especially in tissues with low expression

• Cross-Reactivity: Potential binding to related MATE family transporters in Arabidopsis (which has over 50 MATE genes)

• Post-Translational Modifications: Modifications may affect antibody binding or change apparent molecular weight

• Expression Threshold Detection: Limited sensitivity may prevent detection of low abundance expression

• Sample Preparation Artifacts: Incomplete extraction or protein degradation may affect results

To address these challenges, incorporate quantitative controls, multiple detection methods, and correlation with independent techniques such as RNA expression analysis or functional assays . When presenting data, clearly distinguish between replicated, consistent findings and observations that show variability between experiments.

How should I design experiments to investigate DTX25 function using antibody-based approaches?

Designing comprehensive experiments to investigate DTX25 function requires integration of multiple approaches:

• Expression Profiling: Use DTX25 antibody to determine tissue-specific and development-specific expression patterns

• Subcellular Localization: Combine immunofluorescence with organelle markers to determine precise subcellular location

• Stress Response: Monitor DTX25 expression changes under various biotic and abiotic stresses

• Protein Interactions: Use co-immunoprecipitation with DTX25 antibody to identify interaction partners

• Functional Correlation: Correlate protein levels with phenotypic observations in wild-type vs. mutant plants

This multi-faceted approach provides complementary lines of evidence about DTX25 function. When designing time-course experiments, consider both short-term responses (minutes to hours) and long-term adaptations (days to weeks) to various stimuli . Integrate antibody-based findings with genetic and physiological data for comprehensive functional characterization.

What future research directions are promising for DTX25 antibody applications?

Several emerging research directions could expand the utility of DTX25 antibody in plant biology research:

• Systems Biology Integration: Combining DTX25 protein expression data with transcriptomics, metabolomics, and phenomics

• Evolutionary Conservation Studies: Using DTX25 antibody to examine conservation of MATE transporters across plant species

• Environmental Adaptation: Investigating DTX25's role in plant responses to changing environmental conditions

• Crop Improvement Applications: Translating DTX25 functional insights from Arabidopsis to crop species

• Method Development: Optimizing immunoprecipitation protocols for DTX25 to enable protein complex studies