At5g04720 Antibody

What is the At5g04720 protein and what is its role in plant immunity?

At5g04720 encodes ADR1-L2 (Activated Disease Resistance 1-Like 2), a coiled-coil NLR-type (CC-NLR) immune receptor in Arabidopsis thaliana. It belongs to the RPW8-containing helper NLR family that plays critical roles in plant innate immunity . ADR1-L2 is one of three described ADR1 homologs (ADR1, ADR1-L1, and ADR1-L2) that function as helper NLRs in plant defense signaling cascades .

The protein contains:

• RPW8-defined consensus sequences

• Four predicted α-helices with distinct structural properties compared to canonical CNLs

• An N-terminal domain that extends to the P-loop in the NB-ARC domain

ADR1-L2 contributes to basal resistance against pathogens and is involved in effector-triggered immunity signaling networks, making it a valuable target for studying plant disease resistance mechanisms.

What experimental systems are appropriate for At5g04720 antibody use?

At5g04720 antibodies have been validated for several experimental systems, with Western blot being the primary application. Based on available research data:

When working with plant extracts, particularly from Arabidopsis thaliana, protein extraction methods using TCA/acetone followed by denaturation with LDS sample buffer at 70°C for 10 minutes have shown good results in detecting plant proteins of similar nature .

How should I select the appropriate At5g04720 antibody format for my research?

Selection depends on your specific experimental needs. At5g04720 antibodies are available in several formats targeting different regions of the protein:

For initial characterization, a combination of antibodies targeting different regions is recommended to ensure detection regardless of potential protein processing or modification events . For specific applications like studying protein-protein interactions, epitope accessibility should be considered when selecting antibody format.

What validation methods should be employed before using At5g04720 antibodies?

Proper validation is critical for ensuring antibody specificity and reliability:

• Western blot analysis with positive controls:

  • Use extracts from Arabidopsis thaliana wild-type plants

  • Include recombinant At5g04720 protein as positive control

  • Use knockout/knockdown lines as negative controls

• Cross-reactivity assessment:

  • Test antibody against related ADR1 family members (ADR1, ADR1-L1)

  • Evaluate potential cross-reactivity with other NLR proteins

  • Confirm specificity in protein extracts from related plant species if intended for cross-species studies

• Epitope mapping:

  • Determine the exact binding site using synthetic peptides

  • For antibody combinations, deconvolution of individual monoclonal antibodies may be necessary

  • Epitope determination services are available at $100 per combination

• Functional validation:

  • Confirm antibody utility in immunoprecipitation experiments

  • Verify antibody effect on protein function if used in functional studies

What are the optimal conditions for Western blot detection of At5g04720?

Based on protocols used for similar plant proteins in Arabidopsis research:

Sample Preparation:

• Extract proteins from fresh tissue using TCA/acetone method

• Use 10-15 μg of total protein per lane

• Denature samples with LDS sample buffer at 70°C for 10 minutes

Electrophoresis and Transfer:

• Use 12% SDS-PAGE for optimal separation

• Transfer to PVDF membrane (0.2 μm pore size) using semi-dry transfer

• Expected molecular weight: ~40-45 kDa

Immunodetection:

• Block membrane with 5% milk in TBS-T overnight at 4°C

• Primary antibody dilution: 1:1000 in TBS-T for 2 hours at room temperature

• Wash three times for 15 minutes each in TBS-T

• Use appropriate HRP-conjugated secondary antibody

• Develop using standard ECL detection methods

This protocol has been effective for detecting similar plant proteins like HY5 and may require minor adjustments for At5g04720.

How can I optimize protein extraction to improve At5g04720 detection?

Protein extraction methods significantly impact the detection of plant immune receptors:

• Tissue selection and treatment:

  • Young, actively growing tissue typically yields better results

  • Consider inducing defense responses before extraction to increase protein abundance

  • Flash-freeze harvested tissue immediately in liquid nitrogen

• Extraction buffer optimization:

  • Include protease inhibitors to prevent degradation

  • Add phosphatase inhibitors if studying phosphorylation status

  • Consider detergent selection carefully (TRITON X-100, SDS, or NP-40)

• Extraction protocol comparison:

  Method	Advantages	Disadvantages	Recommendation
  TCA/Acetone	Good for removing interfering compounds	May cause protein aggregation	Best for total protein extraction
  Phenol extraction	High purity	Time-consuming	For samples with high lipid content
  Native extraction	Preserves protein activity	Lower yield	For functional studies

• Post-extraction processing:

  • Centrifuge at high speed (>14,000 × g) to remove insoluble material

  • Consider concentration methods if protein abundance is low

  • Avoid repeated freeze-thaw cycles of extracted proteins

What are the key considerations for maintaining At5g04720 antibody stability and activity?

Proper handling of antibodies is essential for experimental success:

• Storage and reconstitution:

  • Store lyophilized antibody at -20°C

  • Reconstitute with the recommended volume of sterile water

  • Make small aliquots to avoid repeated freeze-thaw cycles

  • Spin tubes briefly before opening to collect material from caps

• Working dilution preparation:

  • Prepare fresh working dilutions shortly before use

  • Use high-quality, filtered buffers

  • If preparing stock solutions, include carrier proteins (e.g., BSA) to prevent adsorption

• Long-term stability:

  • Monitor antibody performance over time with consistent positive controls

  • Document lot-to-lot variations

  • Consider adding preservatives (e.g., sodium azide at 0.05%) for long-term storage

  • Avoid exposing antibodies to extreme temperatures or pH conditions

How should I troubleshoot weak or nonspecific signals when using At5g04720 antibodies?

Common issues and solutions:

Additionally, consider signal enhancement systems if protein abundance is low. For plant proteins, tyramide signal amplification has been effective in detecting low-abundance immune receptors.

Can At5g04720 antibodies be used for studying protein-protein interactions in plant immunity?

At5g04720 (ADR1-L2) functions within protein complexes in plant immunity, making interaction studies valuable:

• Co-immunoprecipitation (Co-IP):

  • Use crosslinking agents to stabilize transient interactions

  • Consider native versus denaturing conditions based on interaction strength

  • Pre-clear lysates thoroughly to reduce non-specific binding

  • Include appropriate negative controls (non-specific antibodies, knockout lines)

• Proximity labeling approaches:

  • Consider fusion proteins with BioID or APEX2 for in vivo interaction studies

  • Validate expression and functionality of fusion proteins

  • Optimize labeling conditions for plant tissues

• Interaction verification:

  • Confirm interactions with reciprocal Co-IP experiments

  • Validate with orthogonal methods (Y2H, BiFC)

Research on NLR-type receptors has shown that the N-terminal fragments of CNL receptors interact frequently but rarely self-associate , suggesting potential heteromeric interactions that could be studied using these approaches.

What approaches can be used to study differential expression of At5g04720 during plant immune responses?

Understanding expression dynamics is crucial for characterizing immune receptor function:

• Transcriptional analysis:

  • RT-qPCR for temporal expression patterns

  • RNA-seq for genome-wide expression context

  • In situ hybridization for tissue-specific localization

• Protein level analysis:

  • Western blotting with At5g04720 antibodies for protein abundance

  • Immunohistochemistry for spatial distribution

  • Mass spectrometry for absolute quantification

• Promoter activity:

  • Reporter gene fusions (GUS, GFP) to study promoter activity

  • Chromatin immunoprecipitation to identify transcriptional regulators

• Environmental and pathogen response:

  • Monitor expression changes following pathogen challenge

  • Evaluate expression under different abiotic stresses

  • Compare expression patterns in different genetic backgrounds

This approach has been valuable for understanding the regulation of other plant immune receptors and can provide insights into the role of ADR1-L2 in defense signaling networks.

How does the performance of At5g04720 antibodies compare with antibodies against other plant NLR proteins?

When designing experiments, it's important to understand how At5g04720 antibodies perform relative to other plant immunity protein antibodies:

The reliability of plant immunity antibodies can vary significantly based on protein abundance and extraction methods. For At5g04720 and similar NLR proteins, optimization of extraction protocols is often necessary for consistent results.

What methodological adaptations are needed when using At5g04720 antibodies in different plant species?

While At5g04720 antibodies are primarily designed for Arabidopsis thaliana, researchers may need to adapt methods for studies in other species:

• Cross-reactivity assessment:

  • Test antibody reactivity with recombinant proteins from target species

  • Perform sequence alignment to predict epitope conservation

  • Consider designing species-specific antibodies for divergent sequences

• Extraction modification:

  • Adjust buffer composition based on species-specific compounds

  • For species with high phenolic content, add PVPP to extraction buffer

  • Optimize protein:detergent ratios for membrane-associated proteins

• Detection protocol adaptation:

  • Adjust antibody concentration based on cross-reactivity strength

  • Consider longer incubation times for weaker interactions

  • Use more sensitive detection systems for low-abundance proteins

• Validation in new species:

  • Include appropriate positive and negative controls

  • Perform peptide competition assays to confirm specificity

  • Consider using genetic tools (RNAi, CRISPR) to validate antibody specificity