ATL34 Antibody

What is the ATL34 protein and what is its function in plants?

ATL34 (AT1G35330) is a RING-H2 finger protein belonging to the Arabidopsis thaliana ATL (Arabidopsis Toxicos en Levadura) family of E3 ubiquitin ligases. Like other members of the ATL family, ATL34 likely functions in the ubiquitin-proteasome system, which is fundamental for protein degradation and cellular regulation in eukaryotes . Based on homology to other characterized ATL proteins such as ATL31, ATL34 may be involved in plant stress responses or nutrient signaling pathways . The ATL family in the Arabidopsis genome comprises 91 members, many of which function in environmental stress adaptation . These proteins typically contain a transmembrane-like hydrophobic region at the N-terminus, a GLD region, a RING-H2 type zinc finger domain, and a non-conserved C-terminal region .

What types of ATL34 antibodies are currently available for research?

Based on available information, researchers can access rabbit polyclonal antibodies against Arabidopsis thaliana ATL34 . These antibodies have been validated for applications including ELISA and Western blotting. The antibodies are produced through antigen-affinity purification methods, ensuring specificity for the target protein . When selecting an antibody for research, it's important to verify its reactivity with Arabidopsis thaliana and confirm the isotype (typically IgG for polyclonal antibodies) to ensure compatibility with secondary detection systems.

What are the recommended applications for ATL34 antibodies?

ATL34 antibodies have been validated for Western blot analysis and ELISA applications . For Western blotting, these antibodies can be used to detect native ATL34 protein expression in plant tissues or recombinant ATL34 proteins. In ELISA assays, the antibodies can quantify ATL34 protein levels or assess protein-protein interactions. Based on studies with related ATL proteins, these antibodies might also be suitable for immunoprecipitation experiments to study ATL34 interactions with other proteins, similar to how ATL31 interactions with 14-3-3 proteins were investigated .

How should I design proper controls when using ATL34 antibodies?

When designing experiments with ATL34 antibodies, multiple controls should be implemented:

• Positive control: Include purified recombinant ATL34 protein (≥85% purity) to confirm antibody binding.

• Negative control: Use samples from ATL34 knockout plants or non-plant tissues to verify specificity.

• Loading control: Employ antibodies against housekeeping proteins (e.g., actin, tubulin) to ensure equal protein loading across samples.

• Secondary antibody control: Include samples treated only with secondary antibody to detect non-specific binding.

• Pre-immune serum control: Compare results with pre-immune serum from the same host species to identify background signals.

For advanced applications such as co-immunoprecipitation, include additional controls such as IgG isotype controls and reciprocal immunoprecipitations.

How can I validate the specificity of ATL34 antibodies against other ATL family members?

Validating antibody specificity against homologous proteins is crucial, especially with the ATL family containing 91 members in Arabidopsis . A comprehensive validation approach should include:

• Sequence comparison analysis: Align ATL34 with closely related ATL proteins to identify unique epitopes that would minimize cross-reactivity.

• Recombinant protein testing: Perform Western blot analysis with recombinant proteins from multiple ATL family members (such as ATL6, ATL11, and ATL31) to assess cross-reactivity.

• Genetic validation: Test antibody reactivity in ATL34 knockout or knockdown plant lines compared to wild-type plants.

• Peptide competition assay: Pre-incubate ATL34 antibody with synthetic peptides corresponding to the immunogen to block specific binding, similar to approaches used with ATL31 phospho-peptides .

• Mass spectrometry validation: Following immunoprecipitation with ATL34 antibody, perform mass spectrometry analysis to confirm the identity of captured proteins.

This multi-faceted approach ensures that observed signals truly represent ATL34 and not related family members.

What are the optimal sample preparation methods for detecting ATL34 in plant tissues?

For optimal detection of ATL34 in plant tissues, consider the following methodological approach:

• Tissue selection: Based on expression patterns of ATL family proteins, focus on tissues where ATL34 is most likely expressed (e.g., young seedlings, leaves under stress conditions) .

• Extraction buffer optimization: Use a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

  • Phosphatase inhibitors (if phosphorylation is of interest)

• Membrane protein enrichment: Since many ATL proteins contain transmembrane-like domains , include membrane fractionation steps:

  • Homogenize tissue in buffer without detergents

  • Centrifuge at 10,000 × g to remove debris

  • Ultracentrifuge supernatant at 100,000 × g

  • Resuspend membrane pellet in buffer with detergents

• Protein denaturation: Heat samples at 95°C for 5 minutes in Laemmli buffer with reducing agent, noting that membrane proteins may require alternative denaturation methods.

• Sample loading: Load 20-50 μg of total protein for whole cell lysates, or 10-20 μg for enriched membrane fractions.

These optimized methods increase the likelihood of successful ATL34 detection while minimizing background interference.

How can I investigate the E3 ubiquitin ligase activity of ATL34 using specific antibodies?

To investigate the E3 ubiquitin ligase activity of ATL34, you can adapt protocols used for other ATL family members such as ATL31 with the following methodological approach:

• In vitro ubiquitination assay:

  • Express and purify recombinant ATL34 (available at ≥85% purity)

  • Combine with E1 enzyme, E2 conjugating enzyme, ubiquitin, ATP, and potential substrates

  • Incubate at 30°C for 1-3 hours

  • Detect ubiquitinated products via Western blot using both anti-ubiquitin and anti-ATL34 antibodies

• Substrate identification:

  • Perform yeast two-hybrid screening to identify potential interacting proteins

  • Validate interactions via co-immunoprecipitation with ATL34 antibodies

  • Test if identified proteins are ubiquitinated in an ATL34-dependent manner

• Mutation analysis:

  • Create point mutations in the RING-H2 domain of ATL34

  • Compare ubiquitination activity between wild-type and mutant proteins

  • Use ATL34 antibodies to confirm expression levels of both wild-type and mutant proteins

• In vivo ubiquitination:

  • Express tagged versions of ATL34 and potential substrates in plants

  • Immunoprecipitate with ATL34 antibodies under denaturing conditions

  • Probe for ubiquitination using anti-ubiquitin antibodies

This comprehensive approach will provide insights into the enzymatic function of ATL34 and identify its potential substrates in plant cells.

What methods can I use to study post-translational modifications of ATL34?

Based on research on related ATL proteins like ATL31, which is regulated by phosphorylation , you can investigate post-translational modifications of ATL34 using these methodological approaches:

• Phosphorylation analysis:

  • Treat plant samples with phosphatase inhibitors during extraction

  • Perform Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Detect with ATL34 antibodies to visualize mobility shifts

  • Follow up with mass spectrometry to identify specific phosphorylation sites

• Identification of modification sites:

  • Perform immunoprecipitation using ATL34 antibodies

  • Subject purified protein to mass spectrometry analysis

  • Search for modifications such as phosphorylation, ubiquitination, or SUMOylation

  • Confirm findings using site-directed mutagenesis of identified residues

• Functional significance testing:

  • Create phospho-mimetic and phospho-deficient mutants of ATL34

  • Express in knockout backgrounds and assess functional complementation

  • Use ATL34 antibodies to confirm expression levels of mutant proteins

  • Examine protein-protein interactions of modified vs. unmodified forms

• Stimulus-dependent modification:

  • Expose plants to various stresses (nutrient limitation, pathogen challenge)

  • Compare ATL34 modification status before and after treatment

  • Correlate modifications with changes in protein activity or stability

This approach will reveal how post-translational modifications regulate ATL34 function, potentially uncovering regulatory mechanisms similar to those observed for ATL31 .

Why might I be detecting multiple bands when using ATL34 antibodies in Western blots?

Multiple bands in Western blots with ATL34 antibodies could result from several factors:

• Post-translational modifications: ATL proteins, including ATL31, undergo phosphorylation and other modifications that alter electrophoretic mobility . Different phosphorylation states may appear as distinct bands.

• Protein degradation products: As E3 ubiquitin ligases, ATL proteins may undergo autoubiquitination leading to partial degradation. To minimize this:

  • Add proteasome inhibitors (MG132) to extraction buffers

  • Include multiple protease inhibitors

  • Maintain samples at 4°C throughout processing

• Alternative splicing variants: Check genomic databases for potential ATL34 splice variants that could produce proteins of different molecular weights.

• Cross-reactivity: The antibody may detect related ATL family proteins. Verify using:

  • Peptide competition assays

  • ATL34 knockout lines as negative controls

  • Recombinant ATL34 protein as a size reference

• Non-specific binding: Optimize blocking conditions using:

  • 5% BSA instead of milk for phosphorylated proteins

  • Longer blocking times (2-3 hours)

  • Higher dilution of primary antibody

Systematically addressing these possibilities will help distinguish true ATL34 signal from artifacts and related proteins.

How can I optimize immunoprecipitation protocols for studying ATL34 protein interactions?

To optimize immunoprecipitation (IP) of ATL34 for protein interaction studies, adapt protocols used successfully with related ATL proteins with these methodological considerations:

• Extraction buffer optimization:

  • Use mild non-ionic detergents (0.5-1% NP-40 or 0.5% Triton X-100)

  • Include 150-300 mM NaCl to reduce non-specific interactions

  • Add protease and phosphatase inhibitors freshly before extraction

  • For membrane-associated proteins like ATL34, consider including 10% glycerol to stabilize membrane proteins

• Antibody coupling strategies:

  • Direct method: Covalently couple ATL34 antibodies to protein A/G beads using crosslinkers

  • Indirect method: Pre-incubate protein extracts with ATL34 antibodies before adding beads

  • Compare both approaches to determine which yields cleaner results

• Crosslinking considerations:

  • For transient interactions, use membrane-permeable crosslinkers (DSP, formaldehyde)

  • Apply to intact tissues before extraction

  • Include controls with crosslinker but no antibody

• Washing optimization:

  • Test gradient of salt concentrations (150-500 mM)

  • Include low concentrations of detergent (0.1-0.5%)

  • Perform 4-6 washes to remove non-specific binders

• Elution methods:

  • Peptide competition: Use synthetic peptides corresponding to the antibody epitope

  • pH elution: Use 0.1 M glycine pH 2.5 followed by immediate neutralization

  • SDS elution: Use SDS sample buffer at 95°C (most stringent)

These optimized methods will help maintain specific ATL34 interactions while minimizing background contaminants.

How can ATL34 antibodies be used to study plant stress responses?

Based on the roles of other ATL family proteins in stress responses , ATL34 antibodies can be employed to investigate plant stress adaptation through these methodological approaches:

• Expression analysis under stress conditions:

  • Subject plants to various stresses (drought, salt, pathogen infection)

  • Harvest tissues at multiple time points

  • Perform Western blot analysis with ATL34 antibodies to track protein expression

  • Correlate ATL34 levels with stress intensity and duration

• Subcellular localization changes:

  • Perform subcellular fractionation of stressed vs. non-stressed tissues

  • Use ATL34 antibodies for Western blot analysis of each fraction

  • Alternatively, use ATL34 antibodies for immunofluorescence microscopy

  • Document relocalization patterns in response to stress stimuli

• Protein-protein interaction networks:

  • Conduct co-immunoprecipitation with ATL34 antibodies from stressed tissues

  • Identify stress-specific interaction partners by mass spectrometry

  • Verify interactions using reciprocal immunoprecipitation

  • Map dynamic changes in ATL34 protein complexes during stress response

• Post-translational modification changes:

  • Analyze phosphorylation or ubiquitination status of ATL34 under stress

  • Use Phos-tag gels or ubiquitin-specific antibodies in combination with ATL34 antibodies

  • Correlate modifications with stress signaling pathways

This comprehensive approach will provide insights into how ATL34 contributes to plant stress adaptation mechanisms, building on knowledge of related ATL proteins .

What approaches can be used to study the relationship between ATL34 and other ATL family members?

To investigate potential functional overlap or interaction between ATL34 and other ATL family proteins, consider these methodological approaches:

• Co-expression analysis:

  • Perform Western blot analysis using antibodies against multiple ATL proteins

  • Compare expression patterns across tissues and developmental stages

  • Identify conditions where multiple ATL proteins are co-expressed

  • Quantify relative expression levels of different ATL family members

• Protein-protein interaction studies:

  • Conduct co-immunoprecipitation experiments with ATL34 antibodies

  • Probe for other ATL family members in the precipitates

  • Perform yeast two-hybrid or BiFC assays to confirm direct interactions

  • Map interaction domains through deletion mutants

• Functional redundancy assessment:

  • Generate single and multiple ATL gene knockout lines

  • Compare phenotypes between single and higher-order mutants

  • Perform complementation experiments with various ATL genes

  • Use ATL34 antibodies to confirm absence of protein in knockout lines

• Substrate specificity analysis:

  • Identify substrates of ATL34 using immunoprecipitation and mass spectrometry

  • Compare with known substrates of other ATL proteins (e.g., 14-3-3 proteins for ATL31)

  • Perform in vitro ubiquitination assays with various ATL proteins and substrates

  • Determine substrate preferences through competition experiments

These approaches will reveal functional relationships within the ATL family and uncover potential compensatory mechanisms when individual ATL proteins are compromised.

How can I use ATL34 antibodies to study its role in plant development and nutrient response?

Building on findings that ATL31 is involved in carbon/nitrogen nutrient response , you can investigate ATL34's role in plant development and nutrient signaling through these methodological approaches:

• Developmental expression profiling:

  • Harvest tissues from different developmental stages

  • Perform Western blot analysis with ATL34 antibodies

  • Create a temporal map of ATL34 expression

  • Correlate protein levels with specific developmental transitions

• Nutrient response experiments:

  • Grow plants under various carbon/nitrogen ratios (similar to ATL31 studies)

  • Monitor ATL34 protein levels via Western blotting

  • Examine post-translational modifications under different nutrient conditions

  • Compare with expression patterns of known nutrient response genes

• Mutant phenotype characterization:

  • Generate ATL34 overexpression and knockout lines

  • Assess phenotypes under normal and nutrient-stress conditions

  • Use ATL34 antibodies to confirm protein levels in transgenic lines

  • Compare with phenotypes of other ATL mutants (e.g., ATL31)

• Target identification in nutrient signaling:

  • Immunoprecipitate ATL34 from plants under different nutrient conditions

  • Identify interacting proteins by mass spectrometry

  • Focus on proteins involved in nutrient sensing or metabolism

  • Verify if identified targets are ubiquitinated in an ATL34-dependent manner

This systematic approach will reveal whether ATL34 functions in nutrient response pathways similar to ATL31 or has distinct developmental roles.

How can I develop phospho-specific antibodies to study ATL34 regulation?

Based on the finding that phosphorylation regulates ATL31's interaction with 14-3-3 proteins , developing phospho-specific antibodies for ATL34 would be valuable. Here's a methodological approach:

• Phosphorylation site prediction and verification:

  • Use bioinformatic tools to predict potential phosphorylation sites in ATL34

  • Look for conserved motifs similar to the identified sites in ATL31 (Thr209, Ser247, Ser270, Ser303)

  • Verify phosphorylation sites experimentally through mass spectrometry

• Phospho-peptide design:

  • Synthesize 10-15 amino acid peptides containing the phosphorylated residue

  • Position the phosphorylated residue centrally in the peptide

  • Include a C-terminal cysteine for conjugation if not present naturally

  • Synthesize both phosphorylated and non-phosphorylated versions

• Immunization strategy:

  • Conjugate phospho-peptides to carrier proteins (KLH or BSA)

  • Immunize rabbits with the conjugated phospho-peptides

  • Use multiple boost immunizations to increase antibody titer

  • Collect serum and evaluate antibody response by ELISA

• Antibody purification:

  • Perform negative selection using non-phosphorylated peptide columns

  • Follow with positive selection using phosphorylated peptide columns

  • Test specificity using peptide competition assays

  • Validate with phosphatase-treated samples as negative controls

• Validation in plant systems:

  • Test antibodies on wild-type plants and phospho-site mutants

  • Verify detection in plants treated with phosphatase inhibitors

  • Compare signals before and after alkaline phosphatase treatment

  • Perform immunoprecipitation followed by mass spectrometry to confirm specificity

This approach would yield valuable tools for studying the phosphorylation-dependent regulation of ATL34 and its potential interactions with signaling partners.

What are the best approaches for studying ATL34 in protein degradation pathways?

To investigate ATL34's role in protein degradation as an E3 ubiquitin ligase, implement these methodological approaches:

• Ubiquitination assay system:

  • Develop an in vitro system with recombinant ATL34 , E1, E2 enzymes, and ubiquitin

  • Test multiple E2 enzymes to identify functional pairs

  • Use Western blotting with ATL34 antibodies to detect autoubiquitination

  • Include controls with catalytically inactive ATL34 mutants

• Substrate identification and validation:

  • Perform differential proteomics comparing wild-type and ATL34 knockout plants

  • Focus on proteins that accumulate in the absence of ATL34

  • Verify direct ubiquitination using recombinant proteins

  • Confirm in vivo using cycloheximide chase assays in the presence or absence of ATL34

• Proteasome-dependent degradation confirmation:

  • Treat plants with proteasome inhibitors (MG132)

  • Analyze accumulation of potential substrates

  • Use ATL34 antibodies to track changes in ATL34 levels (autoregulation)

  • Compare degradation kinetics in wild-type versus ATL34 mutant plants

• Degradation dynamics under stress conditions:

  • Subject plants to various stresses known to affect related ATL proteins

  • Monitor substrate and ATL34 levels using specific antibodies

  • Track ubiquitination status of substrates during stress response

  • Correlate protein degradation with physiological responses

This systematic approach will establish ATL34's role in targeted protein degradation and identify the biological processes regulated through this mechanism.