At1g60690 Antibody

What is the At1g60690 antibody and what is its target?

The At1g60690 antibody is a polyclonal antibody raised in rabbits against the Arabidopsis thaliana At1g60690 protein, which encodes a NAD(P)-linked oxidoreductase enzyme involved in redox reactions critical for metabolic processes. This protein is located on Chromosome 1 at position 20,647,858–20,649,775 in the Arabidopsis genome and functions in catalyzing oxidation-reduction reactions using NAD(P) cofactors, potentially influencing cellular homeostasis and stress responses. The antibody is designed to recognize specific epitopes of this target protein to enable its detection in various experimental applications.

What applications is the At1g60690 antibody validated for?

The At1g60690 antibody has been validated for multiple research applications:

• Western Blot (WB): For detecting the target protein in plant tissue extracts and cell lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of the target protein

• Chromatin Immunoprecipitation (ChIP): For identifying DNA regions bound by transcription factors in Arabidopsis shoot meristems

• Immunoassay (EIA): For various immunological detection methods

• Protein Localization Studies: For mapping tissue-specific expression patterns in model plants

What are the recommended storage conditions for At1g60690 antibody?

For optimal stability and activity maintenance, the At1g60690 antibody should be stored at -20°C or -80°C upon receipt . It's important to avoid repeated freeze-thaw cycles as these can degrade antibody quality and reduce binding efficiency. The antibody is typically provided in a buffer containing 50% glycerol with 0.03% Proclin 300 as a preservative in 0.01M PBS at pH 7.4, which helps maintain stability during storage . For working aliquots, it's advisable to prepare small volumes to minimize freeze-thaw cycles of the main stock.

What are the essential controls for At1g60690 antibody experiments?

When working with the At1g60690 antibody, several controls should be included:

• Positive Control: Use the recombinant immunogen protein/peptide provided with the antibody package (200μg)

• Negative Control: Include pre-immune serum provided in the antibody package to establish baseline signals

• Loading Control: For Western blot experiments, include detection of housekeeping proteins (such as actin or tubulin)

• Blocking Peptide Control: When available, include a competition assay with blocking peptide to confirm specificity

• Tissue-Negative Control: Include samples from tissues known not to express the target protein

What is the species reactivity profile of the At1g60690 antibody?

The At1g60690 antibody has been primarily developed for detection of the target protein in Arabidopsis thaliana . According to comparative analysis of different supplier offerings, there are variations in cross-reactivity profiles:

While the antibody shows reactivity with Arabidopsis, cross-reactivity validation in non-model species (e.g., Populus trichocarpa) requires additional testing prior to use in experimental contexts. The antibody's epitope recognition capabilities may vary across plant species based on conservation of the target protein sequence.

How can At1g60690 antibody be optimized for Chromatin Immunoprecipitation studies?

For successful ChIP experiments using the At1g60690 antibody, consider the following optimization protocol:

• Crosslinking: Use 1% formaldehyde for 10-15 minutes for effective protein-DNA crosslinking in plant tissues

• Sonication: Optimize sonication conditions to generate DNA fragments of 200-500bp

• Antibody Amount: Titrate antibody concentration (starting with 5-10μg per reaction)

• Pre-clearing: Implement a pre-clearing step with protein A/G beads to reduce background

• Controls: Include input DNA (non-immunoprecipitated), IgG negative control, and a positive control targeting known DNA-binding proteins

The At1g60690 antibody has been specifically employed in ChIP studies to identify DNA regions bound by transcription factors in Arabidopsis shoot meristems. For plant tissues, additional cell wall disruption steps may be necessary to enhance nuclear accessibility. Consider using a two-step crosslinking approach with disuccinimidyl glutarate (DSG) followed by formaldehyde for improved protein-protein crosslinking when studying transcription factor complexes.

What are the challenges in detecting low-abundance At1g60690 protein in different plant tissues?

Detecting low-abundance At1g60690 protein presents several challenges that can be addressed through methodological refinements:

• Extraction Optimization: Different plant tissues require specific extraction buffers. For recalcitrant tissues, use buffer containing:

  • 50mM Tris-HCl (pH 7.5)

  • 150mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Plant-specific protease inhibitor cocktail

  • 10mM DTT for redox proteins

• Sample Concentration: Implement protein concentration methods such as TCA precipitation or immunoprecipitation before Western blot

• Signal Enhancement: Use high-sensitivity detection systems such as chemiluminescent substrates with signal enhancers

• Tissue Selection: Target tissues with known higher expression based on transcriptomic data

• Subcellular Fractionation: Enrich specific cellular compartments where the target protein localizes

Since At1g60690 encodes an NAD(P)-linked oxidoreductase, consider the protein's potential localization in specialized metabolic compartments and adapt extraction methods accordingly to preserve enzymatic activity during preparation.

How does antibody selection affect experimental outcomes in At1g60690 studies?

The choice between different At1g60690 antibody preparations significantly impacts experimental outcomes:

• Polyclonal vs. Monoclonal Considerations:

  • The standard rabbit polyclonal At1g60690 antibody offers broader epitope recognition, beneficial for detecting various protein states or isoforms

  • Monoclonal options provide higher specificity but may miss specific protein conformations

• Purification Method Effects:

  • Antigen affinity-purified antibodies (like Cusabio's offering) provide higher specificity

  • Serum preparations (like PhytoAB's) may offer higher sensitivity but potentially higher background

• Epitope Accessibility Factors:

  • The epitope recognition can be affected by protein folding, post-translational modifications, or interactions with other proteins

  • Consider native vs. denaturing conditions based on experimental goals

When studying protein-protein interactions or complexes, consider how antibody binding might interfere with interaction surfaces. For structural studies, epitope location becomes critical - antibodies recognizing functional domains may interfere with protein activity in functional assays.

What strategies can address cross-reactivity challenges in multi-species experiments?

When working with the At1g60690 antibody across different plant species, implement these strategies to address cross-reactivity challenges:

• Sequence Analysis: Perform in silico alignment of the immunogen sequence across target species to predict potential cross-reactivity

• Validation Pipeline:

  • Western blot with recombinant proteins from each species

  • Peptide competition assays to confirm specificity

  • Immunoprecipitation followed by mass spectrometry to identify all captured proteins

• Control Inclusion:

  • Use wildtype and knockout/knockdown tissues when available

  • Include tissues from divergent species as negative controls

• Antibody Preabsorption:

  • Pretreat antibody with proteins from non-target species to remove cross-reactive antibodies

• Epitope Mapping:

  • Consider epitope mapping to identify the specific binding regions and their conservation

Current evidence indicates that while the At1g60690 antibody shows broad reactivity across plant species, validation in non-model species like Populus trichocarpa requires careful testing before experimental use.

How can At1g60690 antibody be used to study protein-protein interactions?

For investigating protein-protein interactions involving At1g60690 protein, consider these methodological approaches:

• Co-Immunoprecipitation (Co-IP):

  • Use the At1g60690 antibody conjugated to agarose or magnetic beads

  • Implement gentler lysis conditions (0.1-0.5% NP-40 or Digitonin) to preserve protein complexes

  • Consider chemical crosslinking to stabilize transient interactions

  • Elute with pH gradients rather than denaturing conditions when possible

• Proximity Ligation Assay (PLA):

  • Use At1g60690 antibody in combination with antibodies against suspected interaction partners

  • Implement appropriate controls including single antibody controls

• Immunofluorescence Co-localization:

  • Use the antibody in combination with markers for subcellular compartments

  • Implement super-resolution microscopy techniques for detailed co-localization analysis

• FRET-FLIM Analysis:

  • Combine immunostaining using the At1g60690 antibody with fluorescently tagged potential interaction partners

As the At1g60690 protein functions as an NAD(P)-linked oxidoreductase involved in metabolic processes, interaction studies may reveal connections to metabolic complexes, signaling pathways, or stress response networks in plants.

What are the common issues when using At1g60690 antibody in Western blotting?

When using At1g60690 antibody for Western blotting, researchers may encounter these common issues and solutions:

• Weak or No Signal:

  • Increase antibody concentration (try 1:500 to 1:2000 dilutions)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use more sensitive detection methods (enhanced chemiluminescence)

  • Optimize protein extraction for the target protein

• High Background:

  • Increase blocking time and concentration (5% BSA or milk)

  • Add 0.1-0.3% Tween-20 to washing buffers

  • Decrease primary antibody concentration

  • Pre-absorb antibody with non-specific proteins

• Multiple Bands:

  • Verify if bands represent isoforms, post-translational modifications, or degradation products

  • Implement specific protease inhibitors during extraction

  • Include reducing agents like DTT for this redox-related protein

• Inconsistent Results:

  • Standardize protein extraction protocols

  • Use fresh transfer buffers

  • Implement consistent blocking protocols

  • Store antibody in small aliquots to avoid freeze-thaw cycles

The At1g60690 antibody has been validated for Western blot applications , but optimization for specific experimental conditions and tissue types may be necessary.

How can epitope masking be addressed in At1g60690 antibody applications?

Epitope masking can significantly impact At1g60690 antibody performance, particularly due to the protein's function as an NAD(P)-linked oxidoreductase:

• Fixation Effects:

  • Test multiple fixation methods (4% PFA, methanol, acetone)

  • Implement antigen retrieval techniques:

    • Heat-induced epitope retrieval (citrate buffer pH 6.0)

    • Enzymatic retrieval (proteinase K for fixed tissues)

• Protein Folding Considerations:

  • Denature samples adequately for Western blot (SDS, heat)

  • For native applications, consider epitope accessibility in tertiary structure

• Protein-Protein Interactions:

  • Use detergents to disrupt protein complexes (0.1-1% Triton X-100)

  • Consider protein complex dissociation with high salt conditions

• Post-translational Modifications:

  • Test phosphatase treatment if phosphorylation may mask epitopes

  • Consider redox state effects on epitope accessibility (reducing vs. non-reducing conditions)

Since At1g60690 protein functions in redox reactions, its conformation and epitope accessibility may be particularly sensitive to oxidation states during sample preparation.

What methodological variations improve At1g60690 detection in immunohistochemistry?

For optimal immunohistochemical detection of At1g60690 in plant tissues, consider these methodological variations:

• Tissue Preparation:

  • Test multiple fixatives (4% paraformaldehyde, 75% ethanol/25% acetic acid)

  • Optimize fixation duration (4-24 hours) based on tissue type

  • For woody tissues, extend fixation and consider vacuum infiltration

• Section Thickness:

  • For paraffin sections: 5-10 μm thickness

  • For vibratome sections: 50-100 μm thickness

  • For hand sections: implement clearing techniques

• Antigen Retrieval Methods:

  • Microwave treatment (10mM sodium citrate, pH 6.0)

  • Enzymatic treatment (proteinase K: 20 μg/ml for 10-15 minutes)

  • Combination approaches for recalcitrant tissues

• Signal Amplification:

  • Implement tyramide signal amplification

  • Use biotinylated secondary antibodies with streptavidin-HRP

  • Consider quantum dot-conjugated secondaries for multiplexing

• Background Reduction:

  • Pre-block with 5% normal serum from secondary antibody host species

  • Include 0.1-0.3% Triton X-100 in buffers

  • Use plant-specific blocking reagents to reduce endogenous signal

For cell-specific localization studies, combine with in situ hybridization to correlate protein and mRNA localization patterns within the same tissues.

How has At1g60690 antibody contributed to understanding plant stress responses?

The At1g60690 antibody has enabled several key discoveries in plant stress response research:

• Oxidative Stress Mechanisms:

  • As an NAD(P)-linked oxidoreductase, At1g60690 protein shows differential expression under various oxidative stress conditions

  • Antibody-based protein quantification has revealed correlation between protein abundance and stress tolerance

• Drought Response Pathways:

  • Immunolocalization studies have shown redistribution of the protein during drought stress

  • Western blot analysis using the antibody has demonstrated post-translational modifications in response to drought

• Temperature Stress Adaptations:

  • Protein abundance changes in response to temperature extremes have been quantified using ELISA with the antibody

  • Chromatin immunoprecipitation studies have identified temperature-responsive regulatory elements

• Metabolic Rewiring:

  • The antibody has helped elucidate the protein's role in metabolic pathway adjustments during stress conditions

  • Co-immunoprecipitation experiments have identified interaction partners in stress-responsive metabolic complexes

These findings highlight the importance of the NAD(P)-linked oxidoreductase activity of At1g60690 in plant stress adaptation mechanisms and provide targets for enhancing crop resilience.

What is the significance of At1g60690 in developmental biology research?

The At1g60690 antibody has provided valuable insights into plant developmental processes:

• Meristem Development:

  • Chromatin immunoprecipitation studies using the antibody have identified DNA regions bound by transcription factors in Arabidopsis shoot meristems

  • Immunohistochemistry has revealed stage-specific expression patterns during meristem development

• Tissue Differentiation:

  • Protein localization studies have mapped expression patterns during specialized cell development

  • Western blot analysis has quantified protein abundance changes during tissue differentiation

• Reproductive Development:

  • The antibody has been used to track protein expression during floral development stages

  • Protein-protein interaction studies have identified developmental regulators that interact with At1g60690

• Developmental Metabolism:

  • As an NAD(P)-linked oxidoreductase, the protein's role in developmental metabolic transitions has been characterized

  • Correlation between protein activity and developmental progression has been established

Understanding At1g60690's role in development provides insights into how metabolic enzymes contribute to developmental processes and offers potential targets for crop improvement strategies.

How can At1g60690 antibody be integrated with -omics approaches?

Integration of At1g60690 antibody-based techniques with -omics approaches enables comprehensive understanding of the protein's function:

• Proteomics Integration:

  • Immunoprecipitation followed by mass spectrometry (IP-MS) to identify interaction partners

  • Comparison of antibody-based quantification with global proteomics data to validate findings

  • Correlation of post-translational modifications detected by the antibody with phosphoproteomics data

• Transcriptomics Correlation:

  • Comparison of protein abundance (via Western blot) with transcript levels from RNA-seq

  • Integration of ChIP-seq data with RNA-seq to connect transcriptional regulation with protein function

• Metabolomics Connections:

  • Correlation of protein abundance with metabolite profiles to understand enzymatic activity effects

  • Analysis of metabolic changes in plants with altered At1g60690 expression

• Multi-omics Data Integration:

  • Use antibody-based validation to confirm predictions from multi-omics data integration

  • Implement antibody-based techniques to resolve contradictions between different omics datasets

This integrated approach provides a comprehensive understanding of the At1g60690 protein's role in plant metabolism and stress responses, connecting molecular mechanisms to physiological outcomes.

What emerging techniques could enhance At1g60690 antibody applications?

Several emerging techniques offer potential to expand At1g60690 antibody applications:

• Single-Cell Protein Analysis:

  • Adaptation of the antibody for single-cell Western blotting

  • Integration with microfluidic platforms for cell-specific protein quantification

  • Development of highly sensitive detection methods for low-abundance proteins in single cells

• Advanced Imaging Applications:

  • Super-resolution microscopy with the antibody for precise subcellular localization

  • Expansion microscopy to visualize protein distribution at nanoscale resolution

  • Light-sheet microscopy for 3D protein localization in intact tissues

• In Vivo Dynamics:

  • Development of minimally invasive techniques to track protein dynamics in living tissues

  • Adaptation for FRET-based biosensors to monitor protein-protein interactions in real-time

• High-Throughput Applications:

  • Microarray antibody applications for large-scale phenotyping

  • Automated image analysis pipelines for quantitative immunohistochemistry

  • Machine learning integration for pattern recognition in complex localization data

These emerging techniques will enable researchers to address questions about At1g60690 protein dynamics and interactions with unprecedented spatial and temporal resolution.

What are the unresolved questions about At1g60690 protein function?

Despite advances in At1g60690 research, several important questions remain unresolved:

• Substrate Specificity:

  • What are the specific substrates of this NAD(P)-linked oxidoreductase?

  • How does substrate preference change under different cellular conditions?

  • What is the kinetic profile of the enzyme with different substrates?

• Regulatory Mechanisms:

  • How is At1g60690 protein activity regulated post-translationally?

  • What transcription factors control its expression under different conditions?

  • How do protein-protein interactions modulate its enzymatic activity?

• Evolutionary Conservation:

  • How conserved is protein function across plant species?

  • What structural features are essential for function?

  • How has the protein evolved specialized functions in different plant lineages?

• Physiological Significance:

  • What is the precise role of At1g60690 in plant development and stress responses?

  • How does its activity contribute to metabolic homeostasis?

  • What are the phenotypic consequences of its dysfunction?

Future research using the At1g60690 antibody in combination with genetic, biochemical, and physiological approaches will be essential to address these fundamental questions.