At4g18823 Antibody

What is the At4g18823 protein and what is its significance in plant biology?

At4g18823 is a protein encoded by the AT4G18823 gene in Arabidopsis thaliana (Mouse-ear cress), a model organism widely used in plant research. Based on current understanding of Arabidopsis defensins, At4g18823 likely belongs to the defensin family, which comprises multifunctional defense peptides that play critical roles in plant immunity. Defensins in Arabidopsis are of particular interest as this model plant has over 300 predicted defensin genes . The At4g18823 protein may be involved in plant defense mechanisms, potentially contributing to resistance against pathogens. Understanding this protein can provide insights into plant immunity pathways and evolutionary adaptations in defense mechanisms.

What are the key specifications of the At4g18823 Antibody?

The At4g18823 Antibody (CSB-PA642596XA01DOA) is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana At4g18823 protein. It is supplied as a liquid in a storage buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4. This antibody has been purified using antigen affinity methods and is of the IgG isotype. The antibody has been validated for ELISA and Western Blot applications specifically for detecting At4g18823 in Arabidopsis thaliana samples . Researchers should note that this antibody is made-to-order with a lead time of 14-16 weeks, which requires planning experiments well in advance.

How does At4g18823 relate to plant defensin classification and structure?

While the search results don't explicitly classify At4g18823, recent research on Arabidopsis defensins provides context for understanding this protein. Plant defensins are classified into two major categories based on precursor organization:

• Class I defensins: Composed of a signal peptide and mature defensin sequence

• Class II defensins: Contain an additional C-terminal prodomain that requires proteolytic cleavage

Research has identified genes like A7REG2 and A7REG4 in Arabidopsis that encode class II defensins expressed in flowers, ovules, and seeds. These defensins maintain the characteristic βαββ structure stabilized by four disulfide bonds following the typical bond pattern between cysteine residues (Cys I-VIII, II-V, III-VI, IV-VII) . If At4g18823 is confirmed as a defensin, understanding its class and structural features would provide insights into its functional properties and evolutionary relationships.

What protocols are recommended for using At4g18823 Antibody in Western Blot analysis?

When using At4g18823 Antibody in Western Blot experiments, researchers should follow these methodological guidelines:

• Sample Preparation: Extract total protein from Arabidopsis thaliana tissues using standard extraction buffers containing protease inhibitors. For defensin proteins, which are typically low molecular weight, use appropriate polyacrylamide gel percentages (15-20%) to achieve optimal separation.

• Electrophoresis and Transfer: After SDS-PAGE separation, transfer proteins to a PVDF or nitrocellulose membrane. For small defensin proteins, optimize transfer conditions to prevent loss of low molecular weight proteins.

• Blocking and Antibody Incubation: Block membrane with 5% non-fat milk or BSA in TBST. Incubate with At4g18823 Antibody (optimal dilution should be determined empirically, starting with 1:1000). Since this antibody is antigen-affinity purified, it should provide good specificity when properly optimized .

• Detection: Use appropriate anti-rabbit IgG secondary antibody conjugated to HRP and detect signals using chemiluminescence.

• Controls: Always include positive and negative controls to validate specificity. Consider using tissues known to express or not express At4g18823, such as flowers, ovules, or seeds if At4g18823 expression pattern is similar to other defensins .

What are the critical considerations for optimizing ELISA assays with At4g18823 Antibody?

When developing ELISA protocols with At4g18823 Antibody, researchers should consider:

• Antibody Titration: Determine optimal primary antibody concentration through systematic titration. Start with a dilution range of 1:500 to 1:5000 and identify the concentration that provides maximum specific signal with minimal background.

• Antigen Preparation: Prepare protein extracts from Arabidopsis tissues under conditions that preserve the native structure of At4g18823. Consider using both denaturing and non-denaturing conditions to determine optimal antigen presentation.

• Assay Validation: Validate the specificity of the assay using competitive inhibition with recombinant At4g18823 protein. This approach mimics methods used with other plant antibodies, such as the A4 antibody developed for influenza virus detection .

• Cross-Reactivity Assessment: Test for potential cross-reactivity with other closely related defensin proteins from Arabidopsis. This is particularly important given the large number of defensin genes in this species .

• Signal Development and Quantification: Optimize substrate incubation times and develop standard curves using recombinant At4g18823 protein for accurate quantification.

What are the recommended storage conditions to maintain antibody activity?

To maintain optimal activity of the At4g18823 Antibody, follow these storage guidelines:

• Short-term Storage: For periods less than one month, store at 4°C.

• Long-term Storage: For periods exceeding one month, store at -80°C.

• Aliquoting: Upon receipt, prepare small working aliquots to avoid repeated freeze-thaw cycles, which can compromise antibody performance.

• Storage Buffer: The antibody is supplied in a buffer containing 50% glycerol, which helps maintain stability during freeze-thaw cycles .

• Handling: When removing from storage, allow the antibody to equilibrate to room temperature before opening to prevent condensation, which could introduce contamination or dilute the antibody.

How can At4g18823 Antibody be used to study potential defensin functions in plant immunity?

The At4g18823 Antibody offers several approaches for investigating defensin functions in plant immunity:

• Expression Profiling: Use the antibody to detect changes in At4g18823 protein levels following pathogen challenge or immune elicitor treatment. This approach can reveal whether At4g18823 is regulated as part of plant defense responses.

• Localization Studies: Employ immunohistochemistry or immunofluorescence techniques to determine the subcellular localization of At4g18823 during immune responses. This can provide insights into its mechanism of action, similar to how other defensins have been characterized.

• Protein-Protein Interaction Studies: Use the antibody for co-immunoprecipitation experiments to identify potential protein partners involved in immune signaling pathways.

• Comparative Analysis: Compare the expression and regulation of At4g18823 with known defensins to establish functional relationships. Research has shown that defensins like A7REG2 and A7REG4 in Arabidopsis have specific expression patterns in reproductive tissues, suggesting specialized functions .

• Structural Analysis Validation: Use the antibody to purify native At4g18823 for structural studies to confirm predicted features like the βαββ formation and disulfide bonding patterns typical of plant defensins .

What approaches can be used to study potential developmental roles of At4g18823 in reproductive tissues?

Based on the expression patterns observed for other defensin genes in Arabidopsis, researchers can use the At4g18823 Antibody to investigate developmental roles using these methodologies:

• Temporal Expression Analysis: Perform Western blot analysis on protein extracts from different developmental stages of flowers, ovules, and seeds to establish the temporal expression pattern of At4g18823.

• Spatial Expression Mapping: Use immunohistochemistry with the At4g18823 Antibody to precisely map the protein's spatial distribution within reproductive tissues.

• Functional Studies: Compare At4g18823 expression in wild-type plants versus developmental mutants affecting flower or seed development to establish potential regulatory relationships.

• Comparative Analysis: Investigate whether At4g18823 undergoes similar subfunctionalization as observed with A7REG2 and A7REG4, which are duplicated defensin genes showing specialized expression in reproductive tissues .

• Protein Modification Analysis: Determine whether At4g18823 undergoes post-translational processing similar to class II defensins, which require proteolytic cleavage of their C-terminal prodomain for activation .

How should researchers analyze and normalize quantitative data from At4g18823 immunodetection experiments?

When analyzing quantitative data from experiments using At4g18823 Antibody, implement these analytical strategies:

• Normalization Strategies:

  • For Western blots: Normalize signal intensity to housekeeping proteins (e.g., actin, tubulin)

  • For ELISA: Use standard curves generated with recombinant At4g18823 protein

  • For tissue samples: Consider normalizing to total protein concentration or tissue weight

• Statistical Analysis:

  • Perform minimum of three biological replicates for statistical validity

  • Use appropriate statistical tests based on data distribution (parametric or non-parametric)

  • Consider using ANOVA with post-hoc tests for multiple condition comparisons

• Control Samples:

  • Include negative controls (tissues not expressing At4g18823)

  • Use positive controls where possible (tissues known to express At4g18823)

  • Consider competitive inhibition controls to validate specificity

• Data Visualization:

  • Present data with appropriate error bars

  • Consider showing representative blot images alongside quantified data

  • Use consistent scaling and presentation formats across experiments

How should researchers address potential cross-reactivity with other defensin proteins?

Cross-reactivity is an important consideration when working with antibodies against members of large protein families like defensins. Researchers should:

• Pre-absorption Controls: Perform pre-absorption tests with recombinant defensin proteins to assess cross-reactivity with related family members.

• Genetic Validation: Use knockout or knockdown lines of At4g18823 to confirm antibody specificity. The absence or reduction of signal in these lines would validate antibody specificity.

• Epitope Analysis: Consider the specific epitope recognized by the antibody. The antigen used to generate the At4g18823 Antibody was the full recombinant protein , so it may recognize multiple epitopes.

• Comparison with Transcript Data: Correlate protein detection results with transcript expression data from RNA-seq or qRT-PCR to confirm expression patterns match.

• Mass Spectrometry Validation: For critical experiments, confirm the identity of immunoprecipitated proteins using mass spectrometry to definitively establish antibody specificity.

What are common technical challenges when working with At4g18823 Antibody and how can they be addressed?

Researchers may encounter several technical challenges when working with At4g18823 Antibody:

• High Background Signal:

  • Increase blocking agent concentration (try 5% BSA instead of milk)

  • Optimize antibody dilution (test more dilute solutions)

  • Increase washing duration and frequency

  • Consider using different detergents in wash buffers (Tween-20, Triton X-100)

• Weak or No Signal:

  • Ensure protein of interest is expressed in the tested tissue

  • Optimize protein extraction methods for small defensin proteins

  • Reduce antibody dilution

  • Increase protein loading

  • Use more sensitive detection systems

• Multiple Bands in Western Blot:

  • Verify if bands represent different forms of the protein (processed vs. unprocessed)

  • Test more stringent washing conditions

  • Optimize primary antibody concentration

  • Consider pre-absorbing antibody with non-specific proteins

• Inconsistent Results Between Experiments:

  • Standardize protocols rigorously

  • Use the same antibody lot when possible

  • Implement more comprehensive positive and negative controls

  • Develop a detailed laboratory standard operating procedure

What methodological adaptations might be needed when comparing At4g18823 expression across different plant tissues?

When comparing At4g18823 expression across different plant tissues, researchers should consider these methodological adaptations:

• Tissue-Specific Extraction Protocols: Different plant tissues require optimized protein extraction methods. For example:

  • Flower tissues may require gentler extraction conditions

  • Seed tissues often need more aggressive extraction due to high protein and lipid content

  • Root tissues may contain compounds that interfere with antibody binding

• Normalization Strategy:

  • Use tissue-appropriate housekeeping proteins that maintain consistent expression

  • Consider using total protein normalization methods like Stain-Free technology

  • Implement loading controls specific to each tissue type

• Sample Preparation Considerations:

  • Account for developmental stage variations

  • Consider environmental growth conditions that might affect expression

  • Document harvest times and conditions

• Statistical Design:

  • Use appropriate statistical methods for comparing across diverse tissue types

  • Consider nested experimental designs that account for both biological and technical variation

  • Implement power analysis to determine adequate sample sizes for each tissue type