ATL29 Antibody

What is ATL29 protein and what is its function in Arabidopsis thaliana?

ATL29 (Arabidopsis Toxicos en Levadura 29) is a RING-H2 finger protein in Arabidopsis thaliana (Mouse-ear cress). As identified in the search results, ATL29 belongs to the RING-finger protein family, which typically functions as E3 ubiquitin ligases in the ubiquitin-proteasome pathway . The protein is encoded by the ATL29 gene and plays roles in plant stress responses and protein degradation pathways.

The protein carries the UniProt accession number O49691 . Structurally, ATL29 contains a characteristic RING-H2 finger domain that coordinates zinc ions and facilitates protein-protein interactions, particularly with E2 ubiquitin-conjugating enzymes. This interaction is crucial for the protein's role in substrate ubiquitination and subsequent degradation.

What are the key specifications of commercially available ATL29 antibodies?

The commercially available ATL29 antibody has the following specifications based on product documentation:

This information helps researchers select the appropriate antibody for their experimental needs .

What are the recommended experimental applications for ATL29 antibody?

The ATL29 antibody has been validated for the following applications:

• Western Blot (WB): The antibody is suitable for detecting ATL29 protein in plant tissue lysates. For optimal results, use a 1:500 to 1:2000 dilution in 5% BSA or non-fat milk in TBST, and incubate overnight at 4°C.

• Enzyme-Linked Immunosorbent Assay (ELISA): The antibody can be used for quantitative detection of ATL29 protein levels. A typical working dilution ranges from 1:1000 to 1:5000, depending on the specific ELISA format.

When designing experiments with this antibody, researchers should verify cross-reactivity with their specific plant species of interest, as the antibody was raised against Arabidopsis thaliana ATL29 .

What controls should be included when using ATL29 antibody?

For rigorous experimental design when using ATL29 antibody, researchers should include:

• Positive Control: Wild-type Arabidopsis thaliana tissue expressing ATL29

• Negative Control: Either:

  • ATL29 knockout/knockdown plant material

  • Primary antibody omission control

  • Pre-immune serum control at the same concentration

• Loading Control: Detection of housekeeping proteins (e.g., actin, tubulin) to ensure equal loading across samples

• Size Marker: To verify the detected band corresponds to the expected molecular weight of ATL29

How can ATL29 antibody be used to study plant stress responses and protein regulation?

ATL29 antibody can be employed in several advanced techniques to investigate plant stress responses:

• Temporal Expression Analysis: Western blotting with ATL29 antibody can monitor changes in ATL29 protein levels during different stress conditions (e.g., drought, salinity, pathogen exposure). This reveals how quickly the plant mobilizes this E3 ligase in response to stress.

• Co-Immunoprecipitation (Co-IP): Using ATL29 antibody for Co-IP followed by mass spectrometry allows identification of ATL29's interaction partners and potential ubiquitination substrates. This approach requires:

  • Crosslinking of protein complexes in planta

  • Cell lysis under non-denaturing conditions

  • Immunoprecipitation with ATL29 antibody

  • SDS-PAGE separation and mass spectrometry analysis

• Chromatin Immunoprecipitation (ChIP): If ATL29 has nuclear functions, ChIP with ATL29 antibody can identify its genomic binding sites or associated chromatin regions.

• Immunolocalization: Using ATL29 antibody for immunofluorescence microscopy can reveal subcellular localization changes during stress responses.

These methodologies help build a comprehensive understanding of ATL29's role in plant stress adaptation mechanisms.

What are the technical considerations for optimizing Western blots with ATL29 antibody?

Optimizing Western blots with ATL29 antibody requires attention to several technical parameters:

• Extraction Buffer Composition:

  • Include 20mM N-ethylmaleimide to inhibit deubiquitinases

  • Add protease inhibitor cocktail to prevent degradation

  • Consider adding phosphatase inhibitors if studying phosphorylation-dependent regulation

• Sample Preparation:

  • Fresh tissue extraction typically yields better results than frozen samples

  • Rapid processing prevents protein degradation

  • Optimize protein concentration (typically 20-50μg total protein per lane)

• Blocking and Antibody Incubation:

  • Test different blocking reagents (5% BSA versus 5% non-fat milk)

  • Optimize primary antibody concentration (1:500 to 1:2000)

  • Extended primary antibody incubation (overnight at 4°C) often improves sensitivity

• Detection System Selection:

  • Enhanced chemiluminescence (ECL) for standard detection

  • Fluorescent secondary antibodies for quantitative analysis

  • Consider amplification steps for low-abundance detection

Following these guidelines helps ensure specific and reproducible detection of ATL29 protein.

How does the sequence and structural homology of ATL family proteins affect antibody specificity?

The ATL (Arabidopsis Toxicos en Levadura) family comprises multiple RING-H2 finger proteins with structural similarities that can impact antibody specificity:

• Sequence Homology Analysis:

  • ATL29 shares conserved domains with other ATL family members (ATL1, ATL20, ATL32, ATL38, ATL44)

  • The RING-H2 finger domain is particularly conserved, creating potential cross-reactivity

• Epitope Selection Considerations:

  • The ATL29 antibody was raised against the full recombinant protein

  • Potential cross-reactivity is minimized through affinity purification, but not eliminated

  • Researchers should validate specificity in their experimental system

• Validation Methods for Specificity:

  • Western blot comparison with other ATL family members

  • Testing with ATL29 knockout/knockdown lines

  • Peptide competition assay with the immunizing antigen

  • Side-by-side testing with multiple ATL29 antibodies targeting different epitopes

• Bioinformatic Prediction:

  • Analyze potential cross-reactive epitopes using sequence alignment tools

  • Predict antigenicity profiles to identify unique regions

Understanding these structural relationships helps researchers interpret experimental results and troubleshoot potential cross-reactivity issues.

What approaches can be used to quantify ATL29 protein expression levels across different experimental conditions?

For precise quantification of ATL29 protein levels, researchers can employ several complementary approaches:

• Quantitative Western Blotting:

  • Use fluorescent secondary antibodies instead of chemiluminescence

  • Include a standard curve of recombinant ATL29 protein

  • Normalize to multiple housekeeping proteins

  • Analyze with appropriate image quantification software

• ELISA-Based Quantification:

  • Develop a sandwich ELISA using the ATL29 antibody

  • Create a standard curve with purified recombinant ATL29

  • This method offers higher throughput than Western blotting

• Mass Spectrometry-Based Approaches:

  • Selected/Multiple Reaction Monitoring (SRM/MRM)

  • Parallel Reaction Monitoring (PRM)

  • These techniques require specialized equipment but offer high specificity and sensitivity

• Correlation with Transcriptomic Data:

  • Compare protein expression (via antibody detection) with mRNA levels

  • This helps identify post-transcriptional regulation mechanisms

These methods allow researchers to accurately track changes in ATL29 protein abundance under various experimental conditions.

How can ATL29 antibody be used in conjunction with other experimental approaches to study protein-protein interactions?

Integrating ATL29 antibody with complementary techniques creates a powerful approach for studying protein interaction networks:

• Proximity Ligation Assay (PLA):

  • Combines ATL29 antibody with antibodies against potential interaction partners

  • Generates fluorescent signals only when proteins are in close proximity (<40nm)

  • Enables visualization of interactions in situ within plant cells

• Bimolecular Fluorescence Complementation (BiFC) Validation:

  • Results from BiFC experiments can be validated with co-immunoprecipitation using ATL29 antibody

  • This multi-method approach strengthens confidence in interaction results

• Yeast Two-Hybrid (Y2H) Follow-up:

  • Interactions identified by Y2H can be confirmed in planta using ATL29 antibody pull-downs

  • This validates that interactions occur in the native cellular environment

• Cross-Linking Immunoprecipitation (CLIP) Protocols:

  • For studying RNA-protein interactions if ATL29 binds RNA

  • Combines UV cross-linking with immunoprecipitation using ATL29 antibody

• Super-Resolution Microscopy:

  • Immunofluorescence with ATL29 antibody in super-resolution microscopy allows visualization of protein co-localization at the nanoscale level

What are the methodological considerations when using ATL29 antibody in plant stress response studies?

When designing plant stress experiments utilizing ATL29 antibody, researchers should consider:

• Temporal Sampling Strategy:

  • Include multiple time points (early response: 15min, 30min, 1hr; late response: 3hr, 6hr, 24hr)

  • This captures both immediate signaling events and downstream regulatory changes

• Stress Treatment Standardization:

  • Precisely control stress application parameters

  • Document plant developmental stage, growth conditions, and treatment details

  • Include gradient treatments to establish dose-response relationships

• Tissue-Specific Analysis:

  • Separately analyze different plant tissues (roots, shoots, leaves, reproductive organs)

  • Consider cell-type specific responses through tissue fractionation or single-cell approaches

• Combined Stresses:

  • Natural environments often present multiple simultaneous stresses

  • Design experiments with combined stresses (e.g., drought+heat, salt+pathogen)

  • Use ATL29 antibody to track protein responses in these complex scenarios

• Data Integration Framework:

  • Correlate protein-level changes (detected by ATL29 antibody) with:

    • Transcriptomic changes (RNA-seq)

    • Post-translational modifications (phosphoproteomics)

    • Metabolic adjustments (metabolomics)

    • Physiological responses (gas exchange, chlorophyll fluorescence)

This comprehensive approach positions ATL29 protein dynamics within the broader stress response network.