At3g50210 Antibody

What is At3g50210 and what function does it serve in Arabidopsis thaliana?

At3g50210 is an enzyme in Arabidopsis thaliana that belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) family, specifically the C23 paralog group. It functions as a homoarginine-6-hydroxylase, catalyzing the C6-hydroxylation of homoarginine, which is followed by spontaneous decay to guanidine and α-aminoadipic semialdehyde (AASA), which subsequently cyclizes to P6C. This enzymatic activity contributes to guanidine production in plants. Among the three Arabidopsis 2-ODD-C23 paralogs (At3g50210, Din11, and At3g49630), At3g50210 displays the highest affinity for homoarginine with a Km of 0.78±0.15 mM and the highest specific activity (Amax = 21 ± 1.7 nmol s–1 mg–1) .

What are the specifications of commercially available At3g50210 Antibody?

At3g50210 Antibody is typically available as a polyclonal antibody raised in rabbits using recombinant Arabidopsis thaliana At3g50210 protein as the immunogen. The antibody is often supplied in liquid form with a storage buffer containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4). It undergoes purification through antigen affinity methods to enhance specificity and is recommended for research applications including ELISA and Western Blot analysis .

How should At3g50210 Antibody be stored to maintain its activity?

For optimal preservation of At3g50210 antibody activity, storage at -20°C or -80°C is recommended upon receipt. Researchers should avoid repeated freeze-thaw cycles as these can deteriorate the antibody's performance. The antibody is typically provided in a stabilizing buffer containing glycerol, which helps maintain its structural integrity during freeze-thaw transitions. When planning experiments, it's advisable to aliquot the antibody into smaller volumes to minimize the number of freeze-thaw cycles and extend its usable lifespan .

What experimental applications can At3g50210 Antibody be used for?

At3g50210 Antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western Blot (WB) applications. In ELISA, the antibody can be used to detect and quantify At3g50210 protein in complex biological samples. For Western Blot applications, the antibody can identify the target protein after separation by gel electrophoresis and transfer to a membrane, providing information about protein expression levels and molecular weight. These applications must include proper controls to ensure accurate identification of the antigen .

What cross-reactivity considerations should researchers be aware of when using this antibody?

The At3g50210 antibody is specifically raised against Arabidopsis thaliana At3g50210 protein. Researchers should be aware that potential cross-reactivity may occur with closely related paralogs such as Din11 and At3g49630, which share sequence homology with At3g50210. When designing experiments, especially in wild-type plants that express all three paralogs, validation steps should be included to confirm specificity. Using tissue or samples from knockout mutants (such as the at3g50210-1 line mentioned in the literature) can serve as negative controls to verify antibody specificity .

How can At3g50210 Antibody be used to investigate enzyme kinetics in guanidine production pathways?

To investigate enzyme kinetics of At3g50210 in guanidine production, researchers can employ the antibody in immunoprecipitation assays to isolate the native enzyme from plant tissues or recombinant systems. The purified enzyme can then be used for in vitro activity assays measuring oxygen consumption at variable substrate concentrations. Based on published data, researchers should optimize assay conditions around pH 7.2, where At3g50210 shows maximal activity .

The reaction products can be analyzed using LC-MS/MS to detect formation of P6C, succinate, and guanidine. Parameters to measure include Km for homoarginine (expected around 0.78±0.15 mM) and specific activity (approximately 21 ± 1.7 nmol s–1 mg–1). Comparison with other 2-ODD-C23 paralogs (Din11 and At3g49630) can provide insights into the relative contributions of these enzymes to guanidine production. The antibody can also be used to quantify enzyme abundance in different experimental conditions via Western blot or ELISA, correlating protein levels with measured activity .

What strategies can be employed to optimize ELISA protocols when using At3g50210 Antibody?

For optimizing ELISA protocols with At3g50210 Antibody, researchers should implement the robust ELISA-R analytical framework described in recent literature. Rather than using a standard curve (which may not be feasible for plant-specific antibodies like At3g50210), researchers should focus on comparative analysis between experimental groups. The optimization process should include:

• Determination of optimal antibody concentration through titration experiments

• Selection of appropriate blocking reagents to minimize background

• Implementation of end-point titer determination and curve-fitting models

• Statistical analysis using R-based methods to ensure robust data interpretation

Researchers should employ technical replicates and appropriate controls, including samples from At3g50210 knockout plants. Data analysis should incorporate cleaning and arranging the dataset in required formats while maintaining the integrity of raw data files. When comparing experimental groups (e.g., wild-type vs. mutant plants), statistical significance should be established using appropriate tests based on data distribution characteristics .

How can Western blot protocols be optimized for At3g50210 detection in plant tissue samples?

For optimal Western blot detection of At3g50210 in plant tissue samples, researchers should first optimize protein extraction by using a buffer containing protease inhibitors to prevent degradation of the target protein. Based on the enzyme's properties, extraction at pH 7.2-7.5 may help maintain protein stability. Since At3g50210 is part of the 2-ODD family requiring Fe²⁺ and 2-oxoglutarate as cofactors, adding metal chelators like EDTA should be carefully considered as they might affect protein conformation and antibody recognition.

For SDS-PAGE separation, a 10-12% acrylamide gel is typically suitable for resolving proteins in the expected molecular weight range. After transfer to nitrocellulose or PVDF membranes, blocking with 5% non-fat dry milk or BSA is recommended. Primary antibody dilution should be empirically optimized, starting with manufacturer recommendations (typically 1:1000 to 1:5000). For signal detection, both chemiluminescence and fluorescence-based methods can be employed, with the latter offering better quantification capabilities. Controls should include samples from At3g50210 knockout plants (at3g50210-1 line) to verify specificity of the detected bands .

How can At3g50210 Antibody be used to investigate protein localization in conjunction with GFP fusion studies?

To investigate At3g50210 localization using both antibody detection and GFP fusion approaches, researchers can employ a complementary strategy that leverages the strengths of both methods. From the literature, we know that At3g50210:GFP fusion protein has been successfully expressed in stable transgenic lines . Researchers can use these established lines for direct visualization of protein localization via confocal microscopy.

The antibody can then be used for validation through immunohistochemistry or immunogold electron microscopy, providing higher resolution localization data. For co-localization studies, researchers can perform double immunolabeling with the At3g50210 antibody and antibodies against known organelle markers. When planning these experiments, it's important to verify that the GFP tag doesn't interfere with protein localization or function by comparing the results with antibody-based detection in wild-type plants.

For quantitative analysis of protein expression in different cellular compartments, researchers can perform subcellular fractionation followed by Western blot analysis using the At3g50210 antibody. This approach can complement the visual data from GFP fusion studies with quantitative measurements of protein abundance in different cellular compartments .

What considerations should be made when using At3g50210 Antibody with mutant Arabidopsis lines?

When using At3g50210 Antibody with mutant Arabidopsis lines, researchers must first confirm the genetic status of their mutant lines. The literature describes several established mutant lines including T-DNA insertion mutants (at3g50210-1 with a T-DNA insertion in the third exon) and CRISPR/Cas9-generated mutants. These lines should be backcrossed at least 3 times to Col-0 wildtype to eliminate potential off-target mutations or additional insertions .

How can At3g50210 Antibody be used to study protein-protein interactions in plant metabolism?

To study protein-protein interactions involving At3g50210, researchers can employ co-immunoprecipitation (Co-IP) assays using the At3g50210 antibody. This technique allows for the isolation of protein complexes containing At3g50210 from plant tissue extracts under native conditions. The precipitated complexes can then be analyzed by mass spectrometry to identify interacting partners.

For validation of specific interactions, researchers can use techniques such as bimolecular fluorescence complementation (BiFC) or Förster resonance energy transfer (FRET). These approaches require generating fusion constructs with fluorescent protein fragments or fluorophores. The antibody can be used in these experiments for Western blot validation of fusion protein expression.

Researchers studying At3g50210's role in guanidine production should consider investigating potential interactions with enzymes involved in homoarginine biosynthesis or guanidine metabolism. The antibody can also be used to study how these interactions may change under different physiological conditions or in response to environmental stresses, providing insights into the regulation of guanidine production in plants .

What are the common issues encountered when using At3g50210 Antibody in immunological assays?

Common issues when using At3g50210 Antibody in immunological assays include:

• Cross-reactivity with paralogs: Due to sequence similarity between At3g50210 and its paralogs (Din11 and At3g49630), the antibody may cross-react with these related proteins. This can be addressed by including appropriate controls and validation using knockout mutants.

• Variability in plant tissue samples: Plant tissues can contain various compounds that interfere with antibody binding or detection. Optimizing extraction protocols with appropriate buffers and additives can minimize these issues.

• Protein degradation: As an enzyme, At3g50210 may be susceptible to degradation during sample preparation. Adding protease inhibitors and keeping samples cold can help preserve protein integrity.

• Background signal in immunoassays: Non-specific binding can produce high background signals in ELISA or Western blot. Optimizing blocking conditions (using 3-5% BSA or non-fat dry milk) and including appropriate washing steps can reduce background .

How can researchers validate the specificity of At3g50210 Antibody?

To validate At3g50210 Antibody specificity, researchers should implement a multi-step approach:

• Genetic validation: Use samples from At3g50210 knockout plants (at3g50210-1 line) as negative controls in Western blot and ELISA assays. The absence of signal in these samples confirms antibody specificity.

• Peptide competition assay: Pre-incubate the antibody with excess recombinant At3g50210 protein or the specific immunogenic peptide before application to the sample. Disappearance of the signal indicates specificity.

• Cross-reactivity testing: Test the antibody against recombinant proteins of the paralogs (Din11 and At3g49630) to assess potential cross-reactivity.

• Multiple detection methods: Confirm findings using different techniques (ELISA, Western blot, immunohistochemistry) to ensure consistent detection of the target protein.

• Mass spectrometry validation: Immunoprecipitate the protein using the antibody and confirm its identity by mass spectrometry .

What analytical methods can be used to interpret data from At3g50210 Antibody experiments?

For robust analysis of data generated using At3g50210 Antibody, researchers should implement the following analytical approaches:

• ELISA-R methodology: This R-based method provides a framework for robust ELISA data analysis, particularly useful for "non-standard curve" ELISA as might be the case with plant-specific antibodies. It incorporates end-point titer determination and curve-fitting models .

• Western blot quantification: For quantitative Western blot analysis, researchers should use digital image analysis software to measure band intensity. Normalization to loading controls (housekeeping proteins) is essential for accurate quantification.

• Statistical analysis: Apply appropriate statistical tests based on data distribution. For comparing multiple experimental groups, ANOVA followed by post-hoc tests may be appropriate. For time-course experiments, repeated measures ANOVA or mixed models should be considered.

• Validation through multiple approaches: Cross-validate findings using different techniques (e.g., protein levels by Western blot, enzyme activity by biochemical assays, and localization by microscopy) .

Table of Key Properties and Applications of At3g50210 Antibody

How does At3g50210 compare with its paralogs in enzymatic activity?

The three Arabidopsis 2-ODD-C23 paralogs (At3g50210, Din11, and At3g49630) all catalyze the C6-hydroxylation of homoarginine but with different kinetic properties. At3g50210 demonstrates the highest affinity for homoarginine with a Km of 0.78±0.15 mM and the highest specific activity (Amax = 21 ± 1.7 nmol s–1 mg–1). Din11 shows intermediate properties with a Km of 1.9±0.3 mM and specific activity of 13.4 ± 3.2 nmol s–1 mg–1. At3g49630 has the lowest activity with a Km of 4.6±0.2 mM and specific activity of 5.6±1.2 nmol s–1 mg–1 .

These differences in kinetic properties suggest that At3g50210 may play a more significant role in homoarginine metabolism and guanidine production under conditions where substrate availability is limited. When designing experiments to study these enzymes, researchers should consider these kinetic differences and how they might influence the interpretation of results from different genetic backgrounds (single, double, or triple mutants) .

What emerging technologies could enhance the use of At3g50210 Antibody in plant research?

Several emerging technologies could significantly enhance the application of At3g50210 Antibody in plant research:

• Single-cell proteomics: Adapting the antibody for use in single-cell protein profiling could reveal cell-type specific expression patterns of At3g50210, providing insights into its tissue-specific functions.

• Proximity labeling techniques: Combining the antibody with BioID or APEX2 proximity labeling approaches would allow identification of proteins that interact with At3g50210 in their native cellular environment.

• Super-resolution microscopy: Using the antibody in conjunction with techniques like STORM or PALM could provide nanoscale localization information, potentially revealing subcellular microdomains where At3g50210 functions.

• Machine learning approaches: As described in recent literature on antibody research, machine learning methods could be applied to predict antibody-antigen binding characteristics and optimize experimental designs. Active learning strategies could reduce costs by iteratively expanding labeled datasets for training prediction models .

• Domain generalization algorithms: Recent advances in domain generalization could be applied to develop robust predictors for antibody-antigen interactions that maintain performance despite distribution shifts across experimental cycles .