At1g10490 Antibody

What is the At1g10490 protein and why is it studied?

At1g10490 is a gene from Arabidopsis thaliana (Mouse-ear cress) that encodes a protein in the GNAT acetyltransferase family containing a DUF699 domain. The gene is also referred to as T10O24.10 or T10O24_10 in genomic databases . This protein is studied primarily in plant molecular biology research focusing on gene expression regulation and protein modification through acetylation processes. As a putative acetyltransferase, At1g10490 may play roles in various cellular processes including DNA repair, transcriptional regulation, and metabolic pathways.

Understanding acetyltransferases like At1g10490 provides insights into fundamental biological processes in plants. Researchers typically use antibodies against this protein to investigate its expression patterns, subcellular localization, protein-protein interactions, and potential roles in plant development and stress responses.

What detection methods are compatible with At1g10490 antibody?

The At1g10490 polyclonal antibody can be used in several immunological techniques, with Western blotting being the primary application. Based on similar plant antibodies, the recommended dilution for Western blotting is typically around 1:5000 . While the specific At1g10490 antibody data is limited in the search results, comparable plant antibodies can be used in multiple applications including:

• Western blotting (WB) for protein expression analysis

• Immunoprecipitation (IP) for protein-protein interaction studies

• Immunofluorescence (IF) for subcellular localization

• Enzyme-linked immunosorbent assay (ELISA) for quantitative detection

When designing experiments, researchers should validate the antibody for each specific application, as performance can vary significantly between techniques. For example, an antibody that works well in Western blotting may not necessarily perform optimally in immunohistochemistry.

How should At1g10490 antibody be stored and reconstituted?

Proper storage and reconstitution are critical for maintaining antibody functionality. Similar to other plant antibodies like AUX1, the At1g10490 antibody is likely provided in lyophilized form and should be reconstituted according to manufacturer's instructions . Based on standard protocols for similar antibodies, the following guidelines apply:

For lyophilized antibody:

• Store at -20°C in a moisture-free environment

• Reconstitute by adding an appropriate volume of sterile water (typically 50 μl as seen with similar antibodies)

• After reconstitution, make small aliquots to avoid repeated freeze-thaw cycles

• Spin tubes briefly before opening to collect material that might adhere to the cap or sides

For reconstituted antibody:

• Store aliquots at -20°C for long-term storage

• For short-term use (less than one month), storage at 4°C may be acceptable

• Avoid repeated freeze-thaw cycles, as this can degrade the antibody and reduce its efficacy

What controls should be included when using At1g10490 antibody?

When designing experiments with At1g10490 antibody, appropriate controls are essential for result validation. Based on standard antibody protocols, researchers should include:

• Positive control: Recombinant At1g10490 protein, if available, or tissues/cells known to express the target protein

• Negative control: Samples from knockout or knockdown lines of At1g10490, or tissues that do not express the target

• Loading control: Detection of a housekeeping protein (such as actin or tubulin) to normalize protein loading

• Secondary antibody control: Omission of primary antibody to detect potential non-specific binding of secondary antibody

• Peptide competition assay: Pre-incubation of antibody with immunizing peptide to confirm specificity

Including these controls ensures experimental rigor and helps troubleshoot potential issues with antibody specificity or sensitivity.

How can At1g10490 antibody be used to study protein-protein interactions?

The At1g10490 antibody can be utilized in advanced protein interaction studies using techniques like co-immunoprecipitation (Co-IP), proximity ligation assay (PLA), or pull-down assays. These approaches provide insights into protein complex formation and dynamic interactions within cellular contexts.

For co-immunoprecipitation studies:

• Prepare cell/tissue lysates under non-denaturing conditions to preserve protein-protein interactions

• Incubate lysates with At1g10490 antibody to capture the target protein and its interacting partners

• Precipitate antibody-protein complexes using protein A/G beads or other appropriate matrices

• Analyze precipitated proteins by Western blotting, mass spectrometry, or other detection methods

Similar to studies with MTR4 and HEN2 RNA helicases, researchers can use immunoprecipitation followed by mass spectrometry to identify protein complexes containing At1g10490 . This approach has successfully identified novel protein interactions in plant systems, revealing functional associations between proteins involved in similar cellular processes.

What are the best practices for characterizing At1g10490 antibody specificity?

Thorough characterization of antibody specificity is crucial for generating reliable research data. For At1g10490 antibody, researchers should implement a multi-faceted approach to verify specificity:

• Western blot analysis with recombinant At1g10490 protein to confirm recognition of the target at the expected molecular weight

• Testing reactivity in knockout/knockdown lines to confirm signal reduction/elimination

• Mass spectrometry analysis of immunoprecipitated material to confirm target identity

• Cross-reactivity testing against related proteins, particularly other GNAT family acetyltransferases

• Epitope mapping to identify the specific regions recognized by the antibody

Researchers studying plant proteins have employed expression systems like baculovirus-infected insect cells to produce recombinant proteins for antibody validation . For At1g10490, a similar approach could involve expressing the protein in E. coli, yeast, baculovirus, or mammalian cell systems as indicated in the product information .

How can immunolocalization be optimized for At1g10490 in plant tissues?

Optimizing immunolocalization for plant proteins presents unique challenges due to cell wall barriers and tissue-specific fixation requirements. For At1g10490 visualization in Arabidopsis tissues, consider the following methodological adaptations:

• Fixation protocol: Test different fixatives (paraformaldehyde, glutaraldehyde, or combinations) and fixation times to preserve protein epitopes while maintaining tissue morphology

• Cell wall digestion: Incorporate appropriate enzymatic digestion (using cellulase, pectinase, etc.) to improve antibody penetration without disrupting cellular structures

• Antigen retrieval: Evaluate different antigen retrieval methods (heat-induced, enzymatic, or pH-based) to expose masked epitopes

• Signal amplification: Consider tyramide signal amplification or other enhancement methods for detecting low-abundance proteins

• Co-localization: Combine At1g10490 immunolabeling with markers for cellular compartments to determine precise subcellular localization

Researchers have successfully used similar approaches for visualizing plant proteins like AUX1 (auxin transporter) and cell wall components like rhamnogalacturonan I . These studies emphasize the importance of optimizing each step for the specific tissue type and developmental stage being examined.

What strategies can address cross-reactivity issues with At1g10490 antibody?

Cross-reactivity can compromise experimental results when antibodies recognize unintended targets. For At1g10490 antibody, addressing potential cross-reactivity requires systematic evaluation and mitigation strategies:

• Computational analysis: Perform sequence alignment of the immunizing peptide against the Arabidopsis proteome to identify potential cross-reactive proteins

• Experimental validation: Test antibody reactivity against recombinant proteins from related GNAT family members

• Absorption controls: Pre-adsorb antibody with recombinant proteins of suspected cross-reactive targets

• Knockout/knockdown validation: Compare antibody signals in wild-type versus At1g10490 knockout plants to quantify specific versus non-specific signals

• Species specificity: Test reactivity across different plant species to determine conservation of recognition (similar to AUX1 antibody testing in Arabidopsis versus rice)

Addressing cross-reactivity is particularly important for acetyltransferase studies due to the high sequence similarity among family members. Careful validation ensures that observed signals genuinely represent At1g10490 rather than related proteins.

How can inconsistent Western blot results with At1g10490 antibody be resolved?

Inconsistent Western blot results represent a common challenge in antibody-based research. For At1g10490 antibody, troubleshooting should address multiple parameters:

• Sample preparation optimization:

  • Test different extraction buffers (varying detergent types/concentrations, salt concentrations, and pH)

  • Optimize protein denaturation conditions (temperature, reducing agent concentration)

  • Include appropriate protease inhibitors to prevent target degradation

  • Consider native versus denaturing conditions if epitope recognition is affected by protein folding

• Blocking optimization:

  • Compare different blocking agents (milk, BSA, commercial blockers)

  • Adjust blocking time and temperature

  • Test varying concentrations of blocking agent (1-10%)

• Antibody incubation parameters:

  • Evaluate different dilutions of primary antibody (1:1000 to 1:10,000)

  • Test various incubation times and temperatures

  • Consider adding detergents or carrier proteins to reduce background

Similar to optimization procedures for ASK1 antibody or AUX1 antibody , systematic adjustment of these parameters can significantly improve signal-to-noise ratio and reproducibility.

What factors influence the quantitative accuracy of At1g10490 antibody-based assays?

When using At1g10490 antibody for quantitative applications, several factors can influence accuracy and reproducibility:

• Antibody affinity and avidity:

  • Batch-to-batch variation in antibody production can affect binding characteristics

  • Concentration-dependent binding effects, similar to those observed in competitive binding models

  • Epitope accessibility variations in different sample preparations

• Technical considerations:

  • Linearity range for detection (signal may not scale linearly with protein concentration)

  • Detection method sensitivity (chemiluminescence, fluorescence, colorimetric)

  • Image acquisition parameters and quantification software settings

• Sample-related variables:

  • Protein extraction efficiency across different tissues or growth conditions

  • Post-translational modifications affecting epitope recognition

  • Protein stability during sample processing

To address these challenges, researchers should establish standard curves using recombinant At1g10490 protein, implement appropriate normalization controls, and validate quantification across multiple biological replicates.

How do plant-specific compounds affect At1g10490 antibody performance?

Plant tissues contain various compounds that can interfere with immunological techniques. These compounds may particularly affect At1g10490 antibody performance:

• Phenolic compounds and secondary metabolites:

  • Can irreversibly bind to proteins, altering epitope structures

  • May cause non-specific antibody binding or precipitation

  • Can modify proteins through oxidation reactions

• Cell wall components:

  • Polysaccharides like rhamnogalacturonan I can trap antibodies or cause non-specific binding

  • May impede protein extraction or create high background signal

• Abundant plant proteins:

  • RuBisCO and other highly abundant proteins can mask detection of less abundant targets

  • Can cause lane distortion in gel electrophoresis

Mitigation strategies include incorporating polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP) in extraction buffers to remove phenolics, using specialized plant protein extraction kits, and implementing fractionation techniques to enrich for the protein of interest before immunodetection.

How can At1g10490 antibody be used to study acetyltransferase function in plant development?

The At1g10490 antibody provides a valuable tool for investigating the role of this GNAT acetyltransferase in plant developmental processes. Researchers can design comprehensive studies that examine:

• Temporal expression patterns:

  • Track At1g10490 protein levels throughout plant development using Western blotting

  • Compare protein expression with transcript levels to identify post-transcriptional regulation

  • Analyze expression in different tissues and cell types using immunohistochemistry

• Response to environmental stimuli:

  • Monitor protein levels under various stress conditions (drought, salt, pathogen infection)

  • Investigate post-translational modifications that might regulate acetyltransferase activity

  • Examine subcellular relocalization in response to stimuli

• Genetic manipulation studies:

  • Use the antibody to confirm protein depletion in knockout/knockdown lines

  • Validate overexpression lines for functional studies

  • Assess protein levels in lines expressing modified versions of At1g10490

These approaches parallel successful studies of other plant proteins like AUX1, where antibodies have been instrumental in understanding protein function in developmental contexts .

What are the considerations for using At1g10490 antibody in chromatin immunoprecipitation studies?

Given that GNAT acetyltransferases often function in chromatin modification, the At1g10490 antibody could potentially be used in chromatin immunoprecipitation (ChIP) studies to identify genomic targets. Key considerations include:

• Crosslinking optimization:

  • Test different crosslinking agents (formaldehyde, DSG, EGS) and conditions

  • Optimize crosslinking time to capture transient interactions without over-fixation

  • Evaluate dual crosslinking approaches for improved capture efficiency

• Chromatin preparation:

  • Optimize sonication or enzymatic digestion parameters for appropriate fragment size

  • Validate chromatin quality by assessing size distribution and protein-DNA ratios

  • Consider nuclear isolation protocols to enrich for chromatin-associated proteins

• Immunoprecipitation conditions:

  • Test different antibody amounts to determine optimal concentration

  • Evaluate various washing stringencies to balance specificity and sensitivity

  • Include appropriate controls (IgG control, input control, negative genomic regions)

• Target validation:

  • Confirm enrichment of known targets through qPCR before proceeding to genome-wide analyses

  • Validate findings with orthogonal methods (e.g., reporter assays, in vitro binding)

These approaches build upon techniques used for immunoprecipitation studies of other nuclear proteins, as demonstrated in the study of RNA helicases and their associated complexes .

What emerging technologies can enhance At1g10490 antibody-based research?

Recent technological advances offer new opportunities to enhance antibody-based research for proteins like At1g10490:

• Proximity-dependent labeling approaches:

  • BioID or TurboID fusion proteins combined with antibody validation

  • APEX2-mediated proximity labeling for identifying transient interaction partners

  • Integration of these approaches with mass spectrometry for comprehensive interactome mapping

• Single-cell analyses:

  • Adapting antibody-based detection for single-cell protein analysis in plant tissues

  • Combining with single-cell transcriptomics for multi-omics characterization

  • Spatial transcriptomics integration for tissue-contextual understanding

• Advanced imaging techniques:

  • Super-resolution microscopy for precise subcellular localization

  • Live cell imaging using nanobody derivatives of conventional antibodies

  • Multiplexed imaging to simultaneously detect multiple proteins in the same sample

• Automated high-throughput analyses:

  • Robotics-assisted immunoassays for large-scale screening

  • Machine learning approaches for image analysis and pattern recognition

  • Computational modeling of protein interactions similar to antibody binding models

These emerging approaches can significantly enhance the information obtained from At1g10490 antibody-based studies, providing more comprehensive insights into protein function in complex plant systems.

How does At1g10490 antibody reactivity compare across plant species?

Understanding cross-species reactivity is important for comparative studies of GNAT acetyltransferases across plant lineages. Based on patterns observed with other plant antibodies:

Similar to the AUX1 antibody that shows reactivity with Arabidopsis thaliana but not Oryza sativa , the At1g10490 antibody likely has species-specific patterns of reactivity based on epitope conservation. Researchers should empirically test reactivity against target species rather than assuming cross-reactivity based solely on sequence similarity.

How can At1g10490 antibody data be integrated with other -omics datasets?

Integration of antibody-based protein detection data with other -omics approaches provides a more comprehensive understanding of At1g10490 function:

• Transcriptome integration:

  • Compare protein levels detected by At1g10490 antibody with mRNA expression data

  • Identify discrepancies suggesting post-transcriptional regulation

  • Correlate protein expression with transcript levels of interaction partners

• Proteome integration:

  • Combine immunoprecipitation with mass spectrometry to identify interaction networks

  • Compare antibody-based quantification with label-free or labeled quantitative proteomics

  • Analyze post-translational modifications detected in proteomics datasets

• Metabolome integration:

  • Correlate At1g10490 protein levels with metabolites potentially affected by acetyltransferase activity

  • Identify metabolic pathways potentially regulated by At1g10490

  • Analyze metabolic changes in At1g10490 knockout/overexpression lines

• Phenome integration:

  • Connect protein expression patterns with phenotypic data from different genetic backgrounds

  • Develop predictive models connecting protein levels to phenotypic outcomes

  • Establish causative relationships through manipulation of protein levels

This multi-omics integration approach resembles strategies used in complex protein interaction studies, such as the exosome complex analysis that combined immunoprecipitation with mass spectrometry .