At5g43190 Antibody

What is AT5G43190 and what cellular functions does it serve?

AT5G43190 encodes a Galactose oxidase/kelch repeat superfamily protein in Arabidopsis thaliana (thale cress), a model plant organism widely used in molecular biology research . The protein contains kelch repeat domains, which typically mediate protein-protein interactions and are involved in diverse cellular processes. Initially, this protein was investigated as a potentially peroxisomal protein, but subsequent studies using GFP tagging suggested it might be localized to other cellular compartments including mitochondria, cytosol, plastids, or the nucleus . The exact cellular function remains under investigation, though its kelch repeat structure suggests potential roles in protein binding and regulation of cellular processes.

What is the subcellular localization of AT5G43190 protein?

Research indicates that AT5G43190 has been difficult to definitively assign to a single subcellular compartment. According to proteomic studies that separated mitochondrial and peroxisomal fractions, AT5G43190 could not be conclusively assigned to peroxisomes through GFP visualization techniques . The protein may be localized in mitochondria, the cytosol, plastids, or the nucleus based on current evidence. This ambiguity in localization presents challenges for researchers developing subcellular targeting protocols for studying this protein. When designing immunolocalization experiments with AT5G43190 antibodies, researchers should consider multiple potential compartments and use appropriate markers for colocalization studies.

What approaches are recommended for validating AT5G43190 antibodies?

Antibody validation is critical for ensuring experimental reliability. For AT5G43190 antibodies, a comprehensive validation strategy should include multiple complementary techniques. Western blotting should be performed using both wild-type plant tissue and tissues from AT5G43190 knockout/knockdown lines to confirm specificity . Immunoprecipitation followed by mass spectrometry can verify that the antibody captures the intended target. Additionally, researchers should consider using epitope-tagged versions of AT5G43190 to compare detection patterns between the antibody against the native protein and antibodies against the tag. These validation data should be thoroughly documented and shared through antibody data repositories to benefit the wider research community .

What controls are essential when using AT5G43190 antibodies?

When working with AT5G43190 antibodies, several controls are indispensable for ensuring experimental validity. Positive controls should include samples with confirmed AT5G43190 expression, potentially including recombinant AT5G43190 protein or lysates from plants overexpressing the protein. Negative controls should utilize tissues from knockout mutants or plants where AT5G43190 has been silenced via RNA interference. For immunofluorescence experiments, pre-immune serum controls help identify non-specific binding. Additionally, peptide competition assays, where the antibody is pre-incubated with the immunizing peptide, can demonstrate binding specificity. When using secondary antibodies, controls omitting the primary antibody should be included to identify non-specific secondary antibody binding.

What is the recommended protocol for using AT5G43190 antibodies in Western blotting?

For Western blotting with AT5G43190 antibodies, the following protocol is recommended based on standard practices for plant proteins: First, extract proteins from plant tissues using a buffer containing protease inhibitors to prevent degradation. Separate proteins by SDS-PAGE using a 10-12% gel, which provides optimal resolution for the AT5G43190 protein. Transfer proteins to a PVDF or nitrocellulose membrane at 100V for 1 hour using a wet transfer system . Block the membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. Incubate with the primary AT5G43190 antibody at an optimized dilution (typically 1:1000 to 1:5000) overnight at 4°C. Wash thoroughly with TBST, then incubate with HRP-conjugated secondary antibody for 1 hour at room temperature. After washing, detect the signal using chemiluminescence and an imaging system like the Azure Imager .

How can AT5G43190 antibodies be employed in immunolocalization studies?

For immunolocalization of AT5G43190, researchers should consider the following protocol based on techniques described for plant proteins: Fix plant tissues in 4% paraformaldehyde to preserve protein structure and cellular architecture. For root or leaf tissue, embedding in paraffin or resin followed by sectioning is recommended. Alternatively, for cell cultures or protoplasts, direct fixation on slides may be sufficient. Perform antigen retrieval if necessary, followed by blocking with normal serum from the same species as the secondary antibody. Incubate sections with the primary AT5G43190 antibody at optimized dilution (typically 1:100 to 1:500) overnight at 4°C. After washing, apply fluorophore-conjugated secondary antibodies for detection. DAPI counterstaining helps visualize nuclei . Image the samples using an epifluorescence microscope with appropriate filter sets for the chosen fluorophores. When interpreting results, compare with the subcellular markers for mitochondria, peroxisomes, and other organelles to determine precise localization.

How can researchers troubleshoot weak or absent signals when using AT5G43190 antibodies?

When encountering weak or absent signals with AT5G43190 antibodies, systematic troubleshooting approaches are essential. First, verify protein extraction efficiency by examining total protein concentration and using Coomassie staining or Ponceau S to confirm successful protein transfer to membranes. If protein extraction appears adequate, optimize antibody concentration by testing a range of dilutions (e.g., 1:500, 1:1000, 1:2000). Consider extending primary antibody incubation time to overnight at 4°C if shorter incubations yield weak signals. For Western blotting, increasing the amount of loaded protein (up to 50-100 μg) may help detect low-abundance proteins. If the signal remains absent, the protein may be expressed at very low levels under the studied conditions, or post-translational modifications might be interfering with antibody binding. In such cases, try alternative extraction buffers that better preserve protein structure, or consider immunoprecipitation to concentrate the target protein before detection.

How should researchers interpret discrepancies in AT5G43190 detection between different experimental methods?

Discrepancies in AT5G43190 detection across different methods require careful interpretation. If Western blotting shows a band of unexpected size, consider potential post-translational modifications, alternative splicing, or protein degradation. Compare your results with literature reports on AT5G43190 processing. When immunolocalization shows patterns inconsistent with biochemical fractionation data , consider that the protein might shuttle between compartments or that different isoforms might localize differently. If mass spectrometry fails to detect peptides that antibody-based methods identify, evaluate the sample preparation protocols, as different extraction methods may favor certain protein populations. When interpreting contradictory results, construct a comprehensive experimental framework incorporating multiple methods to triangulate the true biological situation. Document all experimental conditions meticulously, as slight variations in sample preparation can significantly impact protein detection.

How can AT5G43190 antibodies be utilized to study protein-protein interactions?

AT5G43190 antibodies can be powerful tools for investigating protein-protein interactions through several sophisticated approaches. Co-immunoprecipitation (Co-IP) using AT5G43190 antibodies can capture the protein along with its interaction partners from plant lysates. The precipitated complexes can then be analyzed by mass spectrometry to identify the interacting proteins. For in situ visualization of protein interactions, proximity ligation assays (PLA) combine AT5G43190 antibodies with antibodies against suspected interaction partners to generate fluorescent signals only when the proteins are in close proximity. Chromatin immunoprecipitation (ChIP) can be performed if AT5G43190 is suspected to interact with DNA or DNA-binding proteins. For confirming direct interactions, researchers can use purified recombinant proteins in pull-down assays followed by detection with AT5G43190 antibodies. When designing these experiments, researchers should consider that the kelch repeat domains in AT5G43190 likely mediate specific protein-protein interactions, and careful buffer optimization may be necessary to preserve these potentially transient interactions.

What approaches can be used to study post-translational modifications of AT5G43190?

Investigating post-translational modifications (PTMs) of AT5G43190 requires specialized techniques. Researchers can use phospho-specific antibodies if phosphorylation sites are known, or employ general phospho-antibodies followed by immunoprecipitation with AT5G43190-specific antibodies. For comprehensive PTM profiling, immunoprecipitate AT5G43190 using validated antibodies and analyze the purified protein by mass spectrometry, which can identify various modifications including phosphorylation, ubiquitination, acetylation, and glycosylation. Two-dimensional gel electrophoresis followed by Western blotting with AT5G43190 antibodies can reveal charge or mass shifts indicative of PTMs. To study the functional significance of identified modifications, compare PTM patterns under different physiological conditions or treatments. The TARGET (Transient Assay Reporting Genome-wide Effects of Transcription factors) system mentioned in the research literature could potentially be adapted to study how transcription factors affect expression or modification of AT5G43190 .

How should researchers design experiments to study AT5G43190 expression under different environmental conditions?

When designing experiments to investigate AT5G43190 expression responses to environmental stimuli, researchers should implement a systematic approach. Begin with a time-course study under various conditions (e.g., drought, salt stress, pathogen exposure, temperature variation) to identify when expression changes occur. Quantitative real-time PCR can provide initial gene expression data, while Western blotting with AT5G43190 antibodies can confirm protein-level changes. Include appropriate housekeeping gene and protein controls that remain stable under the tested conditions. For comprehensive analysis, consider comparing wild-type plants with mutant lines where AT5G43190 is overexpressed or knocked out to understand its functional significance under stress conditions. The experimental design should include biological replicates (minimum n=3) and technical replicates to ensure statistical validity. When possible, complement antibody-based detection with alternative techniques such as GFP-fusion protein visualization or mass spectrometry quantification to provide corroborating evidence for expression changes.

What considerations are important when selecting samples for AT5G43190 antibody-based experiments?

Sample selection and preparation are critical for successful AT5G43190 antibody experiments. Consider developmental stages carefully, as expression may vary significantly throughout plant development. Different tissues (roots, leaves, stems, flowers) should be analyzed separately as subcellular localization and expression levels might differ between tissues. When working with Arabidopsis thaliana, ecotype selection is important as genetic variations can affect protein expression and antibody epitopes. For time-sensitive processes, synchronize plant growth conditions and harvest all samples at the same time of day to control for circadian effects on gene expression. Fresh tissue typically yields better results than frozen samples, though flash-freezing in liquid nitrogen immediately after collection can preserve protein integrity if immediate processing is not possible. Include samples from AT5G43190 mutant lines as controls, such as those available from repositories like the Arabidopsis Biological Resource Center (ABRC) .

What resources are available for researchers working with AT5G43190 antibodies?

Several valuable resources exist for researchers studying AT5G43190. Plasmid repositories like ABRC offer the pENTR-AT5G43190 construct (PENTR-AT5G43190) donated by Joshua Gendron , which can be used to generate expression constructs for recombinant protein production or transgenic plant creation. For antibody selection, researchers should consult antibody data repositories and search engines that compile validation data across multiple applications . These repositories allow researchers to compare antibody performance and select reagents with validated specificity for their experimental system. When published antibodies are unavailable or unsuitable, custom antibody production services can generate antibodies against specific regions of AT5G43190. Computational resources for epitope prediction can guide selection of antigenic regions likely to produce specific antibodies. Researchers should also consider joining collaborative networks or forums dedicated to plant molecular biology techniques, where expertise on working with challenging plant proteins can be shared.