At5g59105 Antibody

What is At5g59105 Antibody and what organism does it target?

At5g59105 Antibody is a polyclonal antibody developed against the At5g59105 protein from Arabidopsis thaliana (Mouse-ear cress). The antibody is produced using a recombinant version of the target protein as an immunogen and is raised in rabbits. It specifically recognizes the At5g59105 protein, which has a UniProt accession number of A8MRC8 . This antibody is designed exclusively for research applications, particularly in plant molecular biology and proteomics studies focusing on Arabidopsis thaliana protein expression and function.

What are the standard validated applications for At5g59105 Antibody?

At5g59105 Antibody has been validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications . These techniques allow researchers to detect and quantify the target protein in different experimental contexts:

• ELISA: Useful for quantitative detection of At5g59105 protein in solution

• Western Blot: Enables detection of the target protein in complex mixtures after separation by gel electrophoresis

When planning experiments, researchers should optimize protocols for their specific sample types, as antibody performance may vary based on sample preparation methods and experimental conditions.

How should At5g59105 Antibody be stored to maintain its efficacy?

For optimal preservation of antibody activity, At5g59105 Antibody should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and reduce binding efficiency. The antibody is supplied in a liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . If frequent use is anticipated, preparing small working aliquots is recommended to minimize freeze-thaw cycles and extend the functional lifespan of the antibody.

What controls are essential when using At5g59105 Antibody in Western blot experiments?

When conducting Western blot experiments with At5g59105 Antibody, the following controls are critical:

• Positive control: Lysate from Arabidopsis thaliana tissue known to express the At5g59105 protein

• Negative control: Lysate from tissue where the target protein is absent or from At5g59105 knockout lines

• Secondary antibody control: Sample incubated with only the secondary antibody to assess non-specific binding

• Loading control: Probing for a housekeeping protein (like actin or tubulin) to normalize protein loading

Additionally, including a pre-adsorption control, where the antibody is pre-incubated with excess recombinant At5g59105 protein before use, can help verify specificity . This approach is particularly valuable when working with new batches of the antibody or when troubleshooting unexpected results.

How can I optimize immunodetection protocols for At5g59105 Antibody?

Optimizing immunodetection with At5g59105 Antibody requires systematic adjustment of several parameters:

When optimizing, change only one parameter at a time and perform controlled comparisons. For challenging samples or low-abundance targets, consider signal amplification systems or more sensitive detection methods. Remember that this antibody has been affinity-purified against the antigen, which typically improves specificity but may require adjusted working concentrations compared to crude antisera .

How can At5g59105 Antibody be used in co-immunoprecipitation studies of protein complexes?

For co-immunoprecipitation (Co-IP) studies utilizing At5g59105 Antibody, researchers should consider:

• Antibody coupling strategy: Direct coupling to solid support (e.g., protein A/G beads) can minimize antibody contamination in eluted samples. Given that At5g59105 Antibody is a rabbit polyclonal, protein A beads would provide efficient capture .

• Lysis conditions: Use a gentle lysis buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate with protease inhibitors) to preserve protein-protein interactions.

• Pre-clearing step: Pre-clear lysates with protein A beads alone to reduce non-specific binding.

• Cross-validation: Confirm interactions through reciprocal Co-IP experiments with antibodies against suspected interacting partners.

• Controls: Include IgG-only controls and lysates from tissues not expressing the target protein.

When analyzing co-immunoprecipitated complexes, mass spectrometry can identify novel interacting partners, providing insights into the functional networks involving the At5g59105 protein in Arabidopsis cellular processes.

What are common sources of false positives/negatives when using At5g59105 Antibody, and how can they be addressed?

When working with At5g59105 Antibody, researchers may encounter several challenges:

Sources of false positives:

• Cross-reactivity with similar proteins: Perform competitive blocking with recombinant At5g59105 protein

• Non-specific secondary antibody binding: Use secondary antibodies with appropriate specificity (anti-rabbit that is F(ab')₂-specific rather than whole IgG)

• Excessive antibody concentration: Titrate antibody to determine optimal working dilution

Sources of false negatives:

• Protein degradation: Add appropriate protease inhibitors during sample preparation

• Epitope masking: Test multiple extraction/denaturation conditions

• Insufficient transfer in Western blots: Validate transfer efficiency with reversible staining

• Low target abundance: Implement signal amplification or enrichment strategies

A systematic approach to troubleshooting involves isolating each variable and testing alternative conditions. When interpreting questionable results, triangulation with orthogonal detection methods (e.g., mass spectrometry or RNA analysis) can provide additional confidence in data interpretation.

How can I distinguish between specific and non-specific signals when my Western blot shows multiple bands?

Multiple bands in Western blots using At5g59105 Antibody may represent specific detection of protein isoforms, post-translational modifications, degradation products, or non-specific binding. To distinguish between these possibilities:

• Compare with predicted molecular weight: The expected band should correspond to the predicted size of At5g59105 protein.

• Validation in knockout/knockdown samples: Test the antibody in samples where At5g59105 is absent or depleted; specific bands should disappear or diminish.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide; specific bands should be blocked while non-specific signals remain.

• Analyze expression patterns: Compare band patterns across tissues with known differential expression of the target.

• Optimize blocking conditions: Test alternative blocking agents (BSA vs. milk) and increased blocking stringency.

• Sequential probing: Strip and reprobe membranes with antibodies against tags if working with tagged versions of the protein.

When analyzing complex band patterns, it's helpful to create a systematic table documenting the molecular weights of observed bands and their presence/absence under various experimental conditions to identify consistent specific signals.

What approaches can optimize immunofluorescence localization studies using At5g59105 Antibody?

Although At5g59105 Antibody has not been explicitly validated for immunofluorescence, researchers interested in subcellular localization studies might consider:

• Fixation optimization: Compare paraformaldehyde (3-4%) with alternative fixatives like methanol or glutaraldehyde to preserve epitope accessibility.

• Antigen retrieval methods: Test heat-induced or enzymatic antigen retrieval if initial staining is weak.

• Signal amplification: Consider tyramide signal amplification or quantum dot conjugates for detecting low-abundance targets.

• Co-localization controls: Include markers for specific subcellular compartments to establish localization patterns.

• Validation approaches:

  • Express fluorescently-tagged At5g59105 and compare with antibody staining

  • Use RNA interference to confirm specificity through signal reduction

When capturing images, standardize exposure settings across samples and controls to enable quantitative comparisons of localization patterns under different experimental conditions.

How can the specificity of At5g59105 Antibody be validated in challenging experimental systems?

Validating antibody specificity is critical, especially when working with complex plant systems. Advanced validation approaches include:

• Expression correlation: Compare protein detection by the antibody with mRNA expression patterns across tissues or developmental stages.

• Genetic validation: Test antibody reactivity in:

  • Knockout/knockdown lines (signal should be absent/reduced)

  • Overexpression lines (signal should be increased)

  • Heterologous expression systems (expression in non-plant cells)

• Epitope mapping: If working with a polyclonal antibody like At5g59105 Antibody, determine which epitopes are recognized using peptide arrays or truncation constructs.

• Cross-species reactivity assessment: Test the antibody against homologous proteins from related plant species to establish specificity boundaries.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody.

This multi-layered validation approach provides robust evidence for antibody specificity and helps identify potential cross-reactivity that might influence data interpretation.

What is known about the function of At5g59105 protein in Arabidopsis thaliana?

The At5g59105 protein (UniProt: A8MRC8) in Arabidopsis thaliana remains relatively understudied compared to many other plant proteins. Based on available information:

• Gene structure: At5g59105 is located on chromosome 5 of the Arabidopsis genome.

• Predicted function: Bioinformatic analyses suggest potential roles in:

  • Plant development processes

  • Stress response pathways

  • Cellular signaling

• Expression patterns: The gene shows differential expression across tissues and developmental stages, with notable changes in response to certain environmental stressors.

Current understanding of At5g59105 function comes primarily from indirect evidence, including:

• Sequence homology with better-characterized proteins

• Co-expression with genes of known function

• Predicted protein domains and structural features

The At5g59105 Antibody represents an important tool for expanding our understanding of this protein's biological role through direct detection and localization studies in plant tissues .

How can At5g59105 Antibody be integrated with other molecular biology techniques in plant research?

At5g59105 Antibody can be integrated with numerous complementary techniques to build a comprehensive understanding of the target protein's function:

• Proteomics integration:

  • Combine immunoprecipitation with mass spectrometry for interactome analysis

  • Use the antibody for targeted protein quantification in complex samples

  • Implement protein array technologies for high-throughput interaction studies

• Functional genomics approaches:

  • Correlate protein levels (detected by the antibody) with transcriptomic data

  • Analyze protein expression in various mutant backgrounds

  • Combine with CRISPR-edited plant lines for structure-function studies

• Cell biology applications:

  • Pair immunodetection with subcellular fractionation to track protein localization

  • Use the antibody in proximity labeling approaches (BioID, APEX) to identify neighboring proteins

  • Integrate with super-resolution microscopy for precise localization studies

• Developmental biology:

  • Track protein expression throughout plant development

  • Analyze tissue-specific expression patterns using immunohistochemistry

  • Study protein dynamics during environmental responses

This integrated approach leverages the specificity of antibody detection while contextualizing findings within broader molecular and cellular frameworks.

How might At5g59105 Antibody be adapted for emerging single-cell proteomic applications?

Emerging single-cell proteomic technologies represent exciting frontiers for plant biology research using At5g59105 Antibody:

• Antibody-based single-cell sorting:

  • Adapting the antibody for fluorescence-activated cell sorting (FACS) by fluorophore conjugation

  • Implementing index sorting to correlate protein expression with transcriptomic profiles

• In situ protein detection:

  • Applying multiplexed ion beam imaging (MIBI) with metal-conjugated antibodies

  • Implementing cyclic immunofluorescence to detect multiple proteins in the same tissue section

• Spatial proteomics applications:

  • Adapting the antibody for spatial transcriptomics platforms that incorporate protein detection

  • Using the antibody in Slide-seq or similar spatial profiling methods

• Microfluidic applications:

  • Incorporating the antibody into microfluidic systems for single-cell western blotting

  • Developing droplet-based antibody detection systems for high-throughput analysis

These applications would require additional validation and potentially modification of the antibody (e.g., conjugation to fluorophores, metals, or enzymes), but they represent promising avenues for understanding At5g59105 protein dynamics at unprecedented resolution.

What considerations are important when designing experiments comparing At5g59105 expression across different Arabidopsis ecotypes or related plant species?

When designing comparative studies of At5g59105 across different genetic backgrounds:

Careful attention to these factors enables robust comparative studies while avoiding artifacts arising from genetic variation or technical limitations in antibody recognition across diverse plant materials.