At1g64890 Antibody

What is the At1g64890 Antibody and what protein does it target?

The At1g64890 Antibody is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana At1g64890 protein. This antibody specifically recognizes the protein encoded by the At1g64890 gene locus located on chromosome 1 of A. thaliana. It is provided in liquid form with a storage buffer containing 50% Glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . The antibody is purified using antigen affinity methods and is designed specifically for research applications in plant molecular biology.

What are the validated applications for At1g64890 Antibody?

The At1g64890 Antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) applications. These techniques allow researchers to detect and quantify the At1g64890 protein in various experimental contexts . While these are the confirmed applications, researchers should conduct preliminary validation when using this antibody for other immunological techniques such as immunohistochemistry, immunofluorescence, or immunoprecipitation.

What is the recommended storage and handling protocol for At1g64890 Antibody?

Upon receipt, the At1g64890 Antibody should be stored at either -20°C or -80°C. It is crucial to avoid repeated freeze-thaw cycles as these can damage antibody structure and functionality . For routine use, small aliquots can be prepared to minimize freeze-thaw events. The antibody is provided in a stabilizing buffer (50% glycerol with preservative) that helps maintain its activity during storage. Always centrifuge the vial briefly before opening to ensure collection of the entire volume and proper homogenization of the solution.

How should I optimize Western blot protocols for At1g64890 Antibody?

For optimal Western blot results with At1g64890 Antibody, consider the following methodological approach:

• Sample preparation: Extract proteins from Arabidopsis tissues using a buffer containing protease inhibitors

• Protein separation: Use 10-12% SDS-PAGE gels for optimal resolution

• Transfer: Transfer proteins to PVDF or nitrocellulose membranes (0.45 μm pore size)

• Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute At1g64890 Antibody 1:500 to 1:2000 in blocking buffer and incubate overnight at 4°C

• Washing: Wash 3-5 times with TBST, 5 minutes each

• Secondary antibody: Use anti-rabbit IgG HRP-conjugated antibody at 1:5000 to 1:10000 dilution

• Detection: Visualize using enhanced chemiluminescence (ECL) reagents

Always include positive and negative controls to validate specificity, particularly when working with complex plant protein extracts.

What controls should be included when using At1g64890 Antibody in experiments?

When designing experiments with At1g64890 Antibody, several controls should be incorporated:

• Positive control: Use protein extracts from wild-type Arabidopsis thaliana known to express At1g64890

• Negative control: Include samples from At1g64890 knockout/knockdown lines where available

• Secondary antibody control: Omit primary antibody to assess non-specific binding of secondary antibody

• Blocking peptide competition: Pre-incubate antibody with excess immunizing peptide to confirm specificity

• Cross-reactivity assessment: Test against proteins from related species to determine specificity boundaries

These controls help establish specificity and validate experimental findings, particularly when publishing results in peer-reviewed journals.

How can I use At1g64890 Antibody in combination with other techniques for comprehensive protein analysis?

The At1g64890 Antibody can be integrated into multi-technique approaches:

• Combine immunoprecipitation with mass spectrometry to identify protein interaction partners

• Pair Western blotting with RT-PCR to correlate protein expression with mRNA levels

• Use immunofluorescence alongside subcellular fractionation to confirm protein localization

• Employ chromatin immunoprecipitation (ChIP) if At1g64890 is suspected to interact with DNA

• Combine with proteomics approaches to study post-translational modifications

This integrative approach provides multi-level validation and generates more comprehensive understanding of At1g64890 protein function in plant biological processes.

What are common issues encountered with At1g64890 Antibody in Western blots and how can they be resolved?

For Western blot applications specifically, the inclusion of a loading control antibody targeting a constitutively expressed plant protein is recommended to normalize expression data.

How can I validate the specificity of At1g64890 Antibody when working with mutant Arabidopsis lines?

To rigorously validate antibody specificity in mutant studies:

• Use genetic knockouts: The most definitive validation comes from testing the antibody against complete knockout lines for At1g64890, where the target protein signal should be absent

• Employ genetic knockdowns: With RNAi or CRISPR-Cas9 reduced expression lines, the signal intensity should correlate with the degree of knockdown

• Overexpression validation: Test against lines overexpressing tagged versions of At1g64890 to confirm recognition

• Heterologous expression: Express the At1g64890 protein in a system naturally lacking the protein (e.g., E. coli) to confirm specific detection

• Immunoprecipitation-mass spectrometry: Verify that the antibody pulls down the correct protein by mass spectrometric analysis

These approaches collectively provide strong evidence for antibody specificity, particularly important when characterizing novel mutant phenotypes.

How does At1g64890 Antibody compare with other plant protein detection systems in terms of sensitivity and specificity?

The polyclonal nature of the At1g64890 Antibody provides certain advantages and limitations compared to other detection systems:

Compared to monoclonal antibodies like LM18, LM19, and LM20 (used for pectic homogalacturonan detection) , the At1g64890 polyclonal antibody recognizes multiple epitopes on the target protein. This potentially increases sensitivity but may decrease specificity. Unlike the highly characterized monoclonal antibodies against plant cell wall components that have been extensively validated across multiple plant species , the At1g64890 Antibody has been specifically raised against and tested with Arabidopsis thaliana proteins.

What methodologies can be employed to study At1g64890 protein interactions in plant immune responses?

For investigating potential roles of At1g64890 in plant immunity, consider these advanced methodological approaches:

• Co-immunoprecipitation with At1g64890 Antibody followed by mass spectrometry to identify interacting proteins during immune responses

• Proximity labeling approaches (BioID or APEX) to capture transient interactions in native contexts

• Bimolecular fluorescence complementation (BiFC) to visualize protein interactions in planta

• Chromatin immunoprecipitation sequencing (ChIP-seq) if At1g64890 functions in transcriptional regulation during immune responses

• Comparative proteomics between infected and non-infected plants to track At1g64890 protein abundance and modification state

These approaches can reveal functional relationships similar to those observed in other plant-pathogen studies, where antibodies have been critical in understanding immune signaling cascades and receptor-ligand interactions .

How can I adapt immunological techniques used with mammalian AT1R antibodies for plant At1g64890 research?

While the At1g64890 Antibody targets a plant protein distinct from mammalian AT1R (Angiotensin II Receptor Type 1), methodological insights from AT1R antibody research can be adapted:

The extensive research on anti-AT1R autoantibodies in human disease demonstrates the importance of functional validation beyond simple binding assays . Similarly, At1g64890 Antibody applications should include both binding detection (Western blot, ELISA) and functional assessments of the protein's activity when possible.

From studies of AT1R in inflammatory and fibrotic processes , we can adapt approaches such as:

• In vitro functional assays to assess whether antibody binding affects protein activity

• Phosphorylation status analysis of downstream signaling molecules

• Cell-based reporter systems to evaluate signaling pathway activation

• Tissue-specific immunohistochemistry to correlate protein localization with physiological responses

These methodological adaptations require careful optimization specific to plant systems but can substantially enhance the depth of At1g64890 protein characterization.

What emerging technologies might enhance At1g64890 Antibody applications in plant research?

Several cutting-edge technologies show promise for expanding At1g64890 Antibody applications:

• Super-resolution microscopy techniques can provide nanoscale visualization of At1g64890 localization beyond conventional immunofluorescence limitations

• Antibody engineering approaches could enhance specificity and sensitivity through techniques like phage display optimization

• Single-cell proteomics combined with At1g64890 immunolabeling could reveal cell-type specific expression patterns

• Spatial transcriptomics paired with protein detection could correlate transcript and protein distribution

• CRISPR-based tagging systems could complement antibody detection for live-cell imaging applications

These emerging approaches offer opportunities to address current limitations in sensitivity and spatial resolution when studying low-abundance plant proteins like At1g64890.

How might research findings using At1g64890 Antibody contribute to understanding plant development and stress responses?

Research using the At1g64890 Antibody could contribute significantly to plant biology by:

• Elucidating protein function through expression pattern analysis across developmental stages

• Tracking protein abundance changes during various abiotic stresses (drought, salt, temperature)

• Investigating post-translational modifications in response to environmental challenges

• Identifying protein-protein interaction networks in stress signaling pathways

• Validating computational predictions of protein function through experimental confirmation