At1g06090 Antibody

What is the At1g06090 protein in Arabidopsis thaliana?

At1g06090 refers to a specific gene locus in Arabidopsis thaliana encoding a protein that has been characterized in various plant biology studies. This protein is documented in the UniProt database under accession number Q9LND9 . The At1g06090 gene is part of the Arabidopsis genome, which serves as a model system for understanding plant molecular biology, development, and responses to environmental stimuli. Like many plant proteins, understanding its function requires specific molecular tools including antibodies designed to detect its presence in experimental systems.

What applications are recommended for At1g06090 antibody?

The At1g06090 antibody has been validated for specific applications including ELISA (Enzyme-Linked Immunosorbent Assay) and Western blotting (WB) . These techniques allow researchers to detect and quantify the At1g06090 protein in plant tissue extracts and cell preparations. When designing experiments, it's crucial to note that this antibody is intended "For Research Use Only" and not for diagnostic or therapeutic applications . Most laboratories employ this antibody at a recommended dilution of 1:1000 for Western blotting, though optimal dilutions may vary depending on sample preparation methods and detection systems.

How should At1g06090 antibody be stored to maintain activity?

Proper storage is critical for maintaining antibody functionality. The At1g06090 antibody is typically supplied in liquid form and should be stored at -20°C or -80°C upon receipt . The antibody is formulated in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . To extend shelf life and prevent activity loss from repeated freeze-thaw cycles, it's advisable to make small aliquots for routine use. When handling the antibody, brief centrifugation of tubes before opening is recommended to collect any material that might adhere to the cap or sides of the tube, similar to practices used with other plant antibodies .

What is the species reactivity of the At1g06090 antibody?

The At1g06090 antibody has been specifically designed and validated to react with Arabidopsis thaliana proteins . While cross-reactivity with proteins from other plant species may occur based on sequence homology, confirmed reactivity has only been established for Arabidopsis thaliana. This specificity makes the antibody particularly valuable for research focused on this model plant system, though researchers working with related species should conduct preliminary tests to confirm cross-reactivity if attempting to use this antibody with non-Arabidopsis samples.

How can I validate At1g06090 antibody specificity in knockout/knockdown experiments?

Validating antibody specificity is crucial for ensuring experimental rigor. For At1g06090 antibody, a comprehensive validation approach should include:

• Comparing Western blot signals between wild-type Arabidopsis and At1g06090 knockout/knockdown mutants. True specific antibodies will show reduced or absent signal in mutant samples.

• Testing pre-absorption with recombinant At1g06090 protein to confirm binding specificity.

• Performing immunoprecipitation followed by mass spectrometry to confirm the identity of pulled-down proteins.

This methodology mirrors approaches used for other Arabidopsis antibodies, such as those against AtSerpin1, where researchers successfully validated antibody specificity using both knockout mutants and transgenic lines . For instance, in studies of plant serpins, which follow analogous validation protocols, researchers utilized T-DNA insertion lines identified in the SALK collections to create knockout mutants, allowing clear demonstration of antibody specificity .

What strategies can improve At1g06090 protein detection in plant tissues with low expression?

When the At1g06090 protein is expressed at low levels, several methodological approaches can enhance detection:

• Sample enrichment: Subcellular fractionation can concentrate the protein compartment where At1g06090 is predominantly located.

• Signal amplification: Using highly sensitive chemiluminescent substrates or amplification systems for Western blotting.

• Immunoprecipitation: Concentrate the protein of interest prior to detection, a method that has proven successful with other Arabidopsis proteins such as BAK1 .

• Alternative sample preparation: Modify extraction buffers with appropriate protease inhibitors to prevent degradation during processing.

For instance, researchers working with BAK1 antibodies in Arabidopsis successfully detected low-abundance proteins by using 2 μl of antibody per 50 μl of Protein G agarose for immunoprecipitation, followed by Western blotting at 1:5000 dilution . Similar enrichment strategies may prove effective for At1g06090 detection.

How can I optimize immunoprecipitation protocols for At1g06090 interaction studies?

Optimizing immunoprecipitation (IP) protocols for At1g06090 protein interaction studies requires careful consideration of several parameters:

• Crosslinking conditions: If transient interactions are expected, chemical crosslinkers may be necessary to stabilize protein complexes.

• Lysis conditions: Buffer composition (detergent type and concentration, salt concentration) should be optimized to maintain protein-protein interactions while ensuring efficient extraction.

• Antibody coupling: Covalently linking the At1g06090 antibody to solid support can minimize co-elution of antibody chains that may interfere with downstream analysis.

• Controls: Include IgG controls and, where possible, samples from At1g06090 knockout plants.

Following precedents from other Arabidopsis protein studies, researchers have successfully used covalently linked antibodies with protein A immunoprecipitation kits for isolating protein complexes, followed by liquid chromatography-nanospray tandem mass spectrometry for interaction partner identification .

What protein extraction methods are optimal for At1g06090 detection in Arabidopsis tissues?

Efficient protein extraction is critical for successful At1g06090 detection. Based on protocols developed for similar Arabidopsis proteins:

• Buffer composition: Use extraction buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail.

• Tissue preparation: Flash-freeze plant tissue in liquid nitrogen and grind to a fine powder while maintaining freezing temperatures to prevent protein degradation.

• Protein fractionation: Consider subcellular fractionation techniques if At1g06090 is known to have specific compartmental localization, similar to approaches used for membrane-associated proteins like BAK1 .

• Sample clarification: Centrifuge extracts at high speed (>14,000 × g) to remove insoluble material before analysis.

For Western blot applications, approximately 90 μg of total protein from Arabidopsis seedling extracts is typically loaded per lane, as demonstrated in studies using other antibodies such as anti-AHB1 .

What controls should be included when using At1g06090 antibody in Western blot experiments?

A rigorous Western blot experiment using At1g06090 antibody should include the following controls:

• Positive control: Recombinant At1g06090 protein or extract from tissues known to express the protein.

• Negative control: Extract from At1g06090 knockout/knockdown plants or tissues known not to express the target.

• Loading control: Probing for constitutively expressed proteins (like actin or tubulin) to ensure equal sample loading.

• Antibody specificity control: Pre-incubation of the antibody with immunizing peptide to demonstrate signal suppression.

When analyzing the results, researchers should expect to observe the At1g06090 protein at its predicted molecular weight, with adjustments for any post-translational modifications that might affect migration patterns during SDS-PAGE.

How can I address non-specific binding issues with At1g06090 antibody?

Non-specific binding is a common challenge when working with antibodies in plant systems. For At1g06090 antibody, consider these approaches:

• Blocking optimization: Test different blocking agents (BSA, non-fat dry milk, casein) at various concentrations to reduce background.

• Antibody dilution optimization: Test a range of dilutions to find the optimal signal-to-noise ratio.

• Washing stringency: Increase washing times or add low concentrations of detergents (0.05-0.1% Tween-20) to washing buffers.

• Pre-adsorption: Incubate the antibody with proteins from species that show cross-reactivity to remove antibodies that bind non-specifically.

This approach has proven effective with other Arabidopsis antibodies, where optimized blocking conditions significantly improved specificity .

What strategies can help distinguish between true protein complexes and artifacts in At1g06090 interaction studies?

When investigating protein interactions involving At1g06090, it's essential to distinguish genuine interactions from artifacts:

• Reciprocal immunoprecipitation: Confirm interactions by performing IP with antibodies against both At1g06090 and the putative interaction partner.

• Size exclusion chromatography: Verify that the proteins co-elute in fractions consistent with the predicted complex size.

• Competition assays: Introduce peptides or proteins that might disrupt specific interactions.

• Crosslinking validation: Apply chemical crosslinking to stabilize transient interactions followed by analysis under non-reducing conditions.

These approaches have been successfully employed in studies of plant protein interactions, such as the interaction between Arabidopsis serpin (AtSerpin1) and the cysteine protease RD21, where researchers fractionated proteins by non-reducing SDS-PAGE and used immunoblotting to detect both the free enzyme and protein complex forms .

How can I quantitatively analyze At1g06090 expression levels across different experimental conditions?

For quantitative analysis of At1g06090 expression:

• Densitometry: Use image analysis software to quantify band intensity in Western blots, normalizing to loading controls.

• Quantitative ELISA: Develop standard curves using recombinant At1g06090 protein for absolute quantification.

• Statistical analysis: Apply appropriate statistical tests (t-test, ANOVA) to determine significant differences between conditions.

• Technical replicates: Include at least three technical replicates for each biological sample to account for assay variability.

For meaningful comparisons between experimental conditions, standardization of protein extraction, handling, and detection protocols is essential to minimize technical variation.

How can At1g06090 antibody be used in immunolocalization studies?

For subcellular localization of At1g06090:

• Tissue preparation: Fix Arabidopsis tissues with paraformaldehyde and embed in paraffin or resin for sectioning, or prepare whole-mount specimens for confocal microscopy.

• Antibody optimization: Determine optimal dilution (typically starting at 1:100-1:500) and incubation conditions.

• Detection systems: Use fluorophore-conjugated secondary antibodies for fluorescence microscopy or enzyme-conjugated antibodies for light microscopy.

• Co-localization studies: Combine with known subcellular markers to precisely determine the protein's location within the cell.

The approach follows methodologies developed for other plant proteins, where such techniques have successfully revealed subcellular distribution patterns .

What experimental approaches can integrate At1g06090 antibody with other molecular techniques for comprehensive functional studies?

To gain comprehensive insights into At1g06090 function, integrate antibody-based approaches with other molecular techniques:

• ChIP-seq studies: If At1g06090 has DNA-binding properties, combine chromatin immunoprecipitation with high-throughput sequencing.

• Proteomics integration: Use immunoprecipitation followed by mass spectrometry to identify interaction partners.

• CRISPR/Cas9 editing: Generate precise modifications to At1g06090 and use antibodies to assess resulting protein expression and localization changes.

• Transcriptome correlations: Correlate protein levels detected by At1g06090 antibody with transcript levels from RNA-seq studies to understand post-transcriptional regulation.

These integrated approaches have been successfully implemented for studies of plant proteins such as AtSerpin1, where researchers combined immunoprecipitation, protein identification by mass spectrometry, and functional analyses of knockout mutants to comprehensively characterize protein function and interactions .