At4g09647 Antibody

What is the At4g09647 Antibody and what target does it recognize?

The At4g09647 Antibody (Product Code: CSB-PA649643XA01DOA) is a polyclonal antibody raised in rabbits that specifically targets the At4g09647 protein in Arabidopsis thaliana (Mouse-ear cress). This antibody recognizes recombinant Arabidopsis thaliana At4g09647 protein, which serves as its immunogen. The antibody is designed specifically for research applications and should not be used for diagnostic or therapeutic purposes . The antibody has been developed through antigen affinity purification methods to ensure specificity and reduced background reactivity in experimental applications.

What are the basic properties and storage conditions for the At4g09647 Antibody?

The At4g09647 Antibody is available in liquid form as a non-conjugated polyclonal IgG antibody. It is supplied in a storage buffer composed of 0.03% Proclin 300 preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . For optimal stability and activity retention, the antibody should be stored at either -20°C or -80°C upon receipt. Researchers should avoid repeated freeze-thaw cycles as this can compromise antibody integrity and performance. The product has a made-to-order lead time of 14-16 weeks, which researchers should factor into experimental planning timelines.

What applications is the At4g09647 Antibody validated for?

The At4g09647 Antibody has been validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications . These applications are fundamental for protein detection and quantification in plant research. In Western Blot applications, the antibody can be used to identify the At4g09647 protein in plant tissue lysates, while ELISA applications allow for more quantitative analysis of protein expression levels. When designing experiments, researchers should consider the following application-specific parameters:

*Note: Optimal dilutions should be determined empirically by each laboratory based on specific experimental conditions.

How does the polyclonal nature of the At4g09647 Antibody affect its application in research?

As a polyclonal antibody, the At4g09647 Antibody consists of a heterogeneous mixture of immunoglobulins that recognize multiple epitopes on the target antigen . This characteristic provides several advantages for researchers:

• Enhanced sensitivity for detection of low-abundance targets

• Greater robustness against minor changes in protein conformation

• Increased tolerance to sample preparation variations

• Ability to recognize the target protein in multiple experimental conditions

How can I optimize the At4g09647 Antibody for immunolocalization studies in Arabidopsis tissue sections?

For researchers extending the application of At4g09647 Antibody to immunolocalization studies, several optimization steps are crucial despite this not being a listed validated application. The process requires careful consideration of fixation, permeabilization, and detection methods:

• Tissue Fixation Protocol:

  • Fix Arabidopsis tissues in 4% paraformaldehyde for 2-4 hours at room temperature

  • Alternative fixatives such as glutaraldehyde may preserve antigen structure but can increase autofluorescence

  • After fixation, wash tissues thoroughly in PBS to remove fixative residues

• Antigen Retrieval Considerations:

  • Heat-mediated antigen retrieval using citrate buffer (pH 6.0) may improve antibody binding

  • Enzymatic retrieval using proteinase K (10 μg/mL) for 10-15 minutes can enhance accessibility

  • Test multiple retrieval methods as the At4g09647 epitope sensitivity is not well-characterized

• Antibody Dilution Series:

  • Begin with a dilution series (1:100, 1:250, 1:500, 1:1000) to determine optimal concentration

  • Include extended incubation times (overnight at 4°C) to enhance sensitivity

  • Use a fluorophore-conjugated secondary antibody at manufacturer's recommended dilution

This methodological approach allows researchers to systematically identify optimal conditions for immunolocalization studies, even for antibodies primarily validated for other applications.

What strategies can be employed to validate the specificity of At4g09647 Antibody in transgenic Arabidopsis lines?

Validating antibody specificity is crucial for reliable experimental outcomes, particularly in transgenic plant systems. For At4g09647 Antibody, a comprehensive validation approach should include:

• Genetic Controls:

  • Use At4g09647 knockout/knockdown lines as negative controls

  • Compare antibody reactivity in wild-type versus mutant tissues

  • Generate overexpression lines to confirm increased signal intensity

• Competitive Inhibition Assays:

  • Pre-incubate the antibody with purified recombinant At4g09647 protein

  • Apply the pre-absorbed antibody to samples in parallel with untreated antibody

  • Specific signal should be significantly reduced in the pre-absorbed condition

• Orthogonal Detection Methods:

  • Correlate protein detection with mRNA expression via RT-qPCR

  • Compare results with GFP-tagged At4g09647 protein localization

  • Combine with mass spectrometry analysis of immunoprecipitated material

• Cross-Reactivity Assessment:

  • Test reactivity against closely related proteins in the same family

  • Evaluate performance in multiple plant tissues with varying expression levels

  • Document any unexpected bands or signals for proper interpretation

This multi-faceted validation strategy helps researchers establish confidence in antibody specificity, especially important when working with previously uncharacterized targets in complex plant systems.

How should sample preparation be optimized for At4g09647 Antibody use in Western blot applications?

Effective sample preparation is critical for successful Western blot detection using the At4g09647 Antibody. The following methodological considerations should guide researchers:

• Extraction Buffer Composition:

  • Use RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS)

  • Include protease inhibitor cocktail to prevent target degradation

  • Add phosphatase inhibitors if phosphorylation status is relevant

  • Consider plant-specific additions such as 2% PVPP to remove phenolic compounds

• Tissue Disruption Protocol:

  • Flash-freeze tissue samples in liquid nitrogen

  • Grind thoroughly to a fine powder while maintaining frozen state

  • Transfer immediately to extraction buffer to prevent protein degradation

  • Homogenize consistently between samples to ensure comparable extraction

• Protein Quantification and Loading:

  • Determine protein concentration using Bradford or BCA assay

  • Load 20-50 μg total protein per lane

  • Include a concentration gradient to establish detection limits

  • Run technical replicates to assess reproducibility

• Transfer and Detection Optimization:

  • Use PVDF membranes for enhanced protein binding

  • Optimize blocking conditions (5% non-fat dry milk or BSA)

  • Incubate with primary antibody (At4g09647) overnight at 4°C

  • Utilize enhanced chemiluminescence detection for highest sensitivity

These methodological considerations help researchers maximize detection sensitivity while minimizing background and non-specific signals when using the At4g09647 Antibody.

What approaches can be used to quantify At4g09647 protein levels across different developmental stages of Arabidopsis?

Quantifying protein expression across developmental stages requires careful experimental design and standardized methodologies. For At4g09647 protein, researchers should consider:

• Sample Collection Strategy:

  • Harvest tissues at precisely defined developmental stages

  • Collect samples at the same time of day to control for circadian effects

  • Process all developmental stages in parallel to minimize batch effects

  • Document phenotypic markers to verify developmental stage

• Quantitative Western Blot Protocol:

  • Include recombinant At4g09647 protein standards at known concentrations

  • Use internal loading controls (e.g., actin, tubulin) for normalization

  • Apply the At4g09647 Antibody at optimized concentration

  • Quantify band intensity using digital image analysis software

• ELISA-Based Quantification:

  • Develop a sandwich ELISA using the At4g09647 Antibody

  • Generate a standard curve with recombinant protein

  • Process all developmental stages on the same plate

  • Calculate protein concentration based on absorbance values

• Data Analysis Approach:

  • Apply appropriate statistical tests for developmental series (e.g., ANOVA)

  • Create normalized expression profiles across developmental stages

  • Consider biological replicates from independent plant generations

  • Correlate protein levels with known developmental markers

This comprehensive approach enables accurate quantification of At4g09647 protein levels throughout Arabidopsis development, providing insights into its potential functional roles.

What are the common causes of non-specific binding when using At4g09647 Antibody, and how can they be mitigated?

Non-specific binding can significantly impact experimental results when using antibodies like At4g09647. Researchers commonly encounter these issues and can address them through systematic troubleshooting:

• Common Sources of Non-Specific Binding:

  • Insufficient blocking of membranes or tissue sections

  • Excessive primary antibody concentration

  • Cross-reactivity with related plant proteins

  • Inadequate washing between incubation steps

  • Degraded or denatured antibody samples

• Mitigation Strategies:

  • Optimize blocking conditions (test 5% milk, 3-5% BSA, or commercial blocking buffers)

  • Titrate antibody concentration using a dilution series

  • Increase number and duration of wash steps

  • Add 0.1-0.5% Tween-20 to wash buffers to reduce hydrophobic interactions

  • Consider pre-absorption with plant extract from knockout lines

• Experimental Controls to Implement:

  • Include secondary antibody-only controls to detect non-specific binding

  • Use tissue from knockout plants as negative controls

  • Include competition assays with recombinant protein

  • Test multiple blocking reagents in parallel

By systematically addressing these factors, researchers can significantly improve signal-to-noise ratio and data reliability when working with the At4g09647 Antibody.

How can epitope masking issues be resolved when the At4g09647 Antibody shows inconsistent detection across different experimental conditions?

Epitope masking represents a significant challenge in antibody-based detection methods and may explain inconsistent results when using the At4g09647 Antibody. This issue can be addressed through several methodological approaches:

• Protein Denaturation Conditions:

  • Test multiple denaturation buffers with varying SDS concentrations

  • Compare reducing (with β-mercaptoethanol) versus non-reducing conditions

  • Evaluate different heating temperatures and durations

  • Consider native versus denatured protein detection methods

• Epitope Accessibility Enhancement:

  • Implement antigen retrieval techniques (heat, pH, enzymatic)

  • Test different fixation protocols that preserve epitope structure

  • Use membrane permeabilization agents for immunofluorescence

  • Consider fragmentation of protein complexes that might obscure the epitope

• Post-Translational Modification Considerations:

  • Determine if phosphorylation, glycosylation, or other modifications affect detection

  • Use phosphatase or glycosidase treatments before antibody application

  • Compare results across tissues with different post-translational modification profiles

  • Document any molecular weight shifts that might indicate modifications

• Buffer Optimization Strategy:

  Buffer Component	Standard Condition	Alternative to Test
  pH	7.4	6.8, 8.0
  Salt (NaCl)	150 mM	100 mM, 300 mM
  Detergent	0.1% Tween-20	0.05% Triton X-100
  Blocking agent	5% BSA	5% milk, commercial blockers
  Incubation temperature	4°C overnight	Room temp 2 hours, 37°C 1 hour

By systematically evaluating these parameters, researchers can identify optimal conditions for epitope accessibility and consistent detection across experiments.

How can the At4g09647 Antibody be applied in protein-protein interaction studies to identify binding partners in Arabidopsis?

Investigating protein-protein interactions using the At4g09647 Antibody requires specialized techniques that leverage its specific binding properties. Researchers can implement the following methodological approach:

• Co-Immunoprecipitation Protocol:

  • Prepare plant lysates under non-denaturing conditions

  • Pre-clear lysate with protein A/G beads to reduce non-specific binding

  • Incubate cleared lysate with At4g09647 Antibody (typically 2-5 μg per mg of total protein)

  • Capture antibody-protein complexes using protein A/G beads

  • Elute bound proteins and analyze by mass spectrometry

• Proximity-Based Labeling Approach:

  • Generate fusion constructs combining At4g09647 with BioID or TurboID

  • Express constructs in Arabidopsis via stable transformation

  • Induce proximity labeling with biotin supplementation

  • Purify biotinylated proteins using streptavidin beads

  • Validate interactions by Western blot using At4g09647 Antibody

• Validation of Identified Interactions:

  • Perform reverse co-immunoprecipitation with antibodies against putative partners

  • Utilize yeast two-hybrid or split-luciferase complementation assays

  • Analyze co-localization by dual immunofluorescence microscopy

  • Compare interaction profiles across different tissues or conditions

This systematic approach enables researchers to build comprehensive protein interaction networks centered around the At4g09647 protein, advancing our understanding of its biological functions in Arabidopsis.

What considerations are important when adapting the At4g09647 Antibody for chromatin immunoprecipitation (ChIP) studies?

While the At4g09647 Antibody is not explicitly validated for ChIP applications, researchers interested in exploring DNA-protein interactions may adapt it following these critical considerations:

• Fixation and Chromatin Preparation:

  • Optimize formaldehyde concentration (1-3%) and fixation time (10-20 minutes)

  • Evaluate sonication conditions to generate DNA fragments of 200-500 bp

  • Verify chromatin fragmentation by agarose gel electrophoresis

  • Reserve a portion of sheared chromatin as input control

• Antibody Evaluation for ChIP Compatibility:

  • Perform preliminary IP experiments to confirm antibody functionality in ChIP buffer conditions

  • Compare ChIP efficiency with different amounts of antibody (2-10 μg per reaction)

  • Include IgG controls and, if available, samples from At4g09647 knockout plants

  • Assess enrichment of known targets via qPCR before proceeding to genome-wide analysis

• ChIP-seq Experimental Design:

  • Prepare biological replicates (minimum of three) for robust statistical analysis

  • Include spike-in controls for normalization across samples

  • Sequence to sufficient depth (≥20 million unique mapped reads)

  • Apply rigorous peak calling algorithms with appropriate false discovery rate control

• Validation of ChIP Results:

  • Confirm enrichment at selected loci by ChIP-qPCR

  • Analyze motif enrichment in identified binding regions

  • Correlate binding sites with gene expression data

  • Perform functional studies of regulated genes to establish biological relevance

By carefully considering these methodological aspects, researchers can potentially extend the utility of the At4g09647 Antibody to chromatin studies, though extensive validation is essential for reliable results.