ATL74 Antibody

What is ATL74 antibody and what is its target protein?

ATL74 antibody is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana ATL74 protein. The target protein (UniProt: Q9LZV8) belongs to the RING-H2 finger protein family, which functions as an E3 ubiquitin ligase involved in protein degradation pathways in Arabidopsis thaliana. The antibody is supplied in liquid form containing 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative . The antibody is purified using antigen affinity chromatography to ensure specificity and minimize cross-reactivity with other proteins.

How should ATL74 antibody be stored to maintain optimal activity?

For optimal preservation of ATL74 antibody activity, storage at -20°C or -80°C is recommended immediately upon receipt. Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and subsequent loss of antibody functionality . When working with the antibody, aliquoting into single-use volumes is recommended, with each aliquot being thawed only once and kept on ice during experiments. The antibody's storage buffer (containing 50% glycerol) helps maintain stability during freezing, but proper aliquoting protocols remain essential for preserving long-term reactivity and experimental reproducibility.

What applications has ATL74 antibody been validated for?

ATL74 antibody has been specifically validated for enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) applications . These techniques allow researchers to detect and quantify the ATL74 protein in plant tissue extracts or recombinant protein preparations. For Western blotting, the antibody can detect both native and denatured forms of the protein, making it versatile for different experimental approaches. While not explicitly validated for other applications such as immunohistochemistry or immunoprecipitation, researchers may need to optimize conditions if attempting these applications.

What is the recommended protocol for using ATL74 antibody in Western blot analysis?

For optimal Western blot results with ATL74 antibody, follow this methodological approach:

• Sample preparation: Extract proteins from Arabidopsis thaliana tissues using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail.

• Protein separation: Load 20-50 μg of protein per lane on a 10-12% SDS-PAGE gel.

• Transfer: Transfer proteins to a PVDF membrane (0.45 μm pore size) using standard wet transfer protocols (100V for 60 minutes).

• Blocking: Block the membrane with 5% non-fat dry milk in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature.

• Primary antibody incubation: Dilute ATL74 antibody at 1:1000 to 1:2000 in blocking solution and incubate overnight at 4°C with gentle agitation.

• Washing: Wash membrane 3-5 times with TBST, 5 minutes each.

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit IgG (1:5000 dilution) for 1 hour at room temperature.

• Detection: Use enhanced chemiluminescence (ECL) substrate for signal development.

This protocol can be optimized based on specific experimental conditions and protein expression levels.

How can researchers optimize ELISA protocols using ATL74 antibody?

For developing a reliable ELISA protocol with ATL74 antibody, consider the following methodological steps:

• Coating: Coat high-binding ELISA plates with capture antibody (1-5 μg/ml) in carbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block with 1-3% BSA in PBS for 1-2 hours at room temperature.

• Sample addition: Add protein samples diluted in blocking buffer with 0.05% Tween-20.

• Detection antibody: Use biotinylated or HRP-conjugated detection antibody.

• Signal development: For colorimetric detection, use TMB substrate and measure absorbance at 450 nm.

Optimization Table for ATL74 Antibody ELISA:

Validation experiments should include positive and negative controls to establish specificity and sensitivity ranges.

What are common technical issues when using ATL74 antibody and how can they be resolved?

When working with ATL74 antibody, researchers may encounter several technical challenges. The following troubleshooting approaches address the most common issues:

• High background in Western blots:

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

  • Add 0.1-0.3% Tween-20 to washing buffer

  • Decrease primary antibody concentration

  • Perform additional washing steps

• Weak or no signal detection:

  • Increase antibody concentration or incubation time

  • Check protein extraction efficiency

  • Verify transfer efficiency using Ponceau S staining

  • Test with positive control samples

  • Ensure target protein isn't degraded during preparation

• Non-specific bands:

  • Increase blocking stringency

  • Use more diluted antibody solution

  • Add competitive blocking with recombinant proteins

  • Try gradient gels to improve separation

• Inconsistent results:

  • Standardize protein extraction methods

  • Control for post-translational modifications

  • Avoid repeated freeze-thaw cycles of antibody

Each of these approaches requires systematic testing to determine optimal conditions for specific experimental setups.

How can researchers validate the specificity of ATL74 antibody in their experimental systems?

Validation of ATL74 antibody specificity is crucial for reliable research outcomes. Recommended validation strategies include:

• Positive and negative control samples:

  • Use wild-type Arabidopsis thaliana tissues as positive controls

  • Use atl74 mutant or knockout lines as negative controls

  • Include tissues from other plant species to test cross-reactivity

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare signal between competed and non-competed antibody

  • Specific signals should be significantly reduced in the competed sample

• Recombinant protein testing:

  • Use purified recombinant ATL74 protein at known concentrations

  • Create a standard curve to verify detection limits

  • Test related proteins to determine cross-reactivity profiles

• Orthogonal validation methods:

  • Compare results with alternative antibodies if available

  • Correlate protein detection with mRNA expression data

  • Use mass spectrometry to confirm band identity in Western blots

These validation approaches provide multiple lines of evidence for antibody specificity and increase confidence in experimental results.

How can ATL74 antibody be utilized for studying protein-protein interactions in ubiquitination pathways?

ATL74 antibody can be employed in several advanced methodological approaches to study protein-protein interactions within ubiquitination pathways:

• Co-immunoprecipitation (Co-IP):

  • Use ATL74 antibody to pull down the target protein complex

  • Identify interacting partners through mass spectrometry or Western blotting

  • Verify interactions with reciprocal Co-IP using antibodies against partner proteins

  • Include appropriate controls: IgG control, input samples, and lysates from knockout plants

• Proximity Ligation Assay (PLA):

  • Combine ATL74 antibody with antibodies against suspected interaction partners

  • Visualize protein-protein interactions in situ with fluorescent probes

  • Quantify interaction signals using automated image analysis

• Chromatin Immunoprecipitation (ChIP) for transcriptional regulation studies:

  • Use ATL74 antibody to investigate if the protein associates with specific DNA regions

  • Analyze ChIP samples by qPCR or sequencing to identify binding sites

  • Correlate with transcriptional data to assess functional significance

• FRET-based interaction studies:

  • Use fluorescently labeled secondary antibodies for ATL74 and potential partners

  • Measure energy transfer as evidence of protein proximity

  • Calculate interaction distances based on FRET efficiency

These methodologies provide complementary approaches to characterize the role of ATL74 in ubiquitin-mediated protein degradation pathways in plants.

What strategies can be employed to study post-translational modifications of ATL74 protein using this antibody?

Investigating post-translational modifications (PTMs) of ATL74 requires sophisticated approaches:

• Phosphorylation analysis:

  • Perform immunoprecipitation with ATL74 antibody followed by phospho-specific staining

  • Combine with phosphatase treatment as control

  • Use mass spectrometry to identify specific phosphorylation sites

  • Develop phospho-site specific antibodies for detailed studies

• Ubiquitination detection:

  • Perform denaturing immunoprecipitation to preserve ubiquitin modifications

  • Probe with anti-ubiquitin antibodies after ATL74 immunoprecipitation

  • Use deubiquitinating enzyme treatments as controls

  • Apply targeted mass spectrometry to identify modified lysine residues

• SUMOylation and other modifications:

  • Adapt immunoprecipitation protocols with modification-specific lysis buffers

  • Use modification-specific antibodies (anti-SUMO, etc.) for detection

  • Apply proteomic approaches to identify modification sites

PTM Analysis Workflow for ATL74:

These approaches enable comprehensive characterization of ATL74's post-translational regulation in plant biological processes.

How should researchers interpret variations in ATL74 protein expression patterns across different plant tissues and developmental stages?

When analyzing ATL74 expression data across tissues and developmental stages, consider these methodological approaches:

• Quantitative analysis framework:

  • Normalize ATL74 signal to appropriate housekeeping proteins (e.g., actin, tubulin)

  • Use relative quantification across samples rather than absolute values

  • Apply statistical analysis (ANOVA, t-tests) to determine significant differences

  • Create expression profiles across developmental stages with appropriate biological replicates

• Tissue-specific expression interpretation:

  • Compare expression profiles to transcriptomic databases for correlation

  • Consider tissue-specific post-translational modifications that may affect antibody binding

  • Analyze subcellular localization in different tissues using fractionation techniques

  • Correlate expression patterns with known developmental and stress-response pathways

• Methodological considerations for developmental studies:

  • Maintain consistent sampling protocols across developmental stages

  • Document environmental conditions that may influence expression

  • Consider diurnal variations in protein expression levels

  • Use time-course experiments to capture dynamic expression changes

• Integration with functional data:

  • Correlate expression patterns with phenotypic observations

  • Compare with expression patterns of known interaction partners

  • Relate to known biological pathways involving E3 ubiquitin ligases

This systematic approach helps distinguish between biologically significant variations and technical artifacts in expression data.

What bioinformatic approaches can complement ATL74 antibody-based experimental data?

Integrating bioinformatic analyses with experimental data from ATL74 antibody studies provides deeper biological insights:

• Sequence analysis and structural prediction:

  • Identify conserved domains and motifs in ATL74 protein

  • Predict protein structure using homology modeling

  • Analyze potential functional sites through conservation analysis

  • Predict PTM sites and compare with experimental findings

• Interactome analysis:

  • Use protein-protein interaction databases to predict ATL74 interaction networks

  • Apply gene ontology (GO) enrichment analysis to identify biological processes

  • Construct network models incorporating experimental interaction data

  • Identify hub proteins and key regulatory nodes

• Comparative genomics:

  • Analyze ATL74 orthologs across plant species

  • Identify conserved regulatory elements in promoter regions

  • Perform phylogenetic analysis to understand evolutionary relationships

  • Compare expression patterns across species when data is available

• Integration with transcriptomics data:

  • Correlate protein levels (detected by ATL74 antibody) with mRNA expression

  • Identify discrepancies that may indicate post-transcriptional regulation

  • Analyze co-expressed genes for functional associations

  • Apply machine learning approaches to predict regulatory relationships

These computational approaches provide a framework for interpreting antibody-based experimental data within broader biological contexts.

How can ATL74 antibody be adapted for emerging plant research technologies?

ATL74 antibody can be strategically integrated into cutting-edge plant research through several methodological adaptations:

• Single-cell protein analysis:

  • Optimize ATL74 antibody for mass cytometry (CyTOF) applications

  • Develop fluorescent-conjugated versions for single-cell Western blotting

  • Adapt for microfluidic antibody-based detection systems

  • Integrate with single-cell proteomics workflows

• Live-cell imaging applications:

  • Fragment antibody preparation for cell-penetrating formats

  • Generate Fab fragments or nanobodies based on ATL74 antibody

  • Develop fluorescently tagged antibody derivatives for in vivo tracking

  • Optimize for super-resolution microscopy techniques

• Integration with CRISPR-based approaches:

  • Combine with CRISPR knockouts for validation studies

  • Use with tagged CRISPR-modified ATL74 for functional studies

  • Apply in screening pipelines for identifying regulatory factors

• High-throughput phenotyping:

  • Adapt for automated immunoassay platforms

  • Develop for plant tissue microarray applications

  • Integrate with robotics-based screening methods

  • Combine with image-based phenotyping technologies

These adaptations position ATL74 antibody research at the forefront of plant molecular biology and functional genomics.

What experimental approaches could resolve current contradictions in ATL74 protein function research?

Several methodological strategies can address existing knowledge gaps and contradictions in ATL74 functional studies:

• Conditional expression systems:

  • Develop inducible ATL74 expression lines

  • Create tissue-specific knockdown/overexpression systems

  • Apply temperature-sensitive or chemically-regulated systems

  • Use these systems with ATL74 antibody to track protein dynamics

• Structure-function relationship studies:

  • Generate domain-specific antibodies to complement the full-length ATL74 antibody

  • Create systematic mutant libraries targeting specific protein regions

  • Combine with in vivo activity assays to correlate structure with function

  • Apply hydrogen-deuterium exchange mass spectrometry for conformational studies

• Comprehensive interactome mapping:

  • Perform systematic yeast two-hybrid or split-ubiquitin screens

  • Apply proximity-dependent biotin identification (BioID) with ATL74 as bait

  • Use quantitative proteomics to identify condition-dependent interactions

  • Develop computational models integrating all interaction data

• Environmental response profiling:

  • Study ATL74 protein levels across diverse stress conditions

  • Analyze post-translational modification patterns under different stimuli

  • Identify condition-specific interaction partners

  • Correlate with phenotypic outcomes in various ecological contexts

These approaches provide a framework for resolving conflicting data and developing a unified model of ATL74 function in plant biology.