ATL21C Antibody

What is ATL21C and what is its biological function?

ATL21C (AT2G46493) is a putative RING-H2 finger protein found in Arabidopsis thaliana (Mouse-ear cress). It belongs to the RING/U-box superfamily protein class . RING finger proteins typically function as E3 ubiquitin ligases in the ubiquitin-proteasome pathway, playing crucial roles in protein degradation, plant development, and stress responses. The specific biological functions of ATL21C are still being characterized, but as a member of the RING-H2 finger protein family, it likely participates in protein ubiquitination and subsequent degradation processes.

What types of ATL21C antibodies are currently available for research?

Based on current information, researchers have access to rabbit polyclonal antibodies against Arabidopsis thaliana ATL21C. The primary example is the "Rabbit anti-Arabidopsis thaliana (Mouse-ear cress) ATL21C Polyclonal Antibody" designed to recognize specific epitopes of the ATL21C protein . These antibodies are valuable tools for studying ATL21C expression, localization, and function in plant tissues.

How does ATL21C differ from ATL proteins studied in human disease research?

It's important to distinguish plant ATL21C from the unrelated ATL (Adult T-cell Leukemia) proteins studied in human disease contexts. In medical research, ATL refers to Adult T-cell Leukemia/Lymphoma, a malignancy caused by human T-cell lymphotropic virus type 1 (HTLV-1) . While both use the "ATL" designation, they represent entirely different biological entities - ATL21C being a plant RING-H2 finger protein , while medical ATL research focuses on T-cell malignancies with distinct molecular characteristics and therapeutic approaches .

What are the recommended protocols for using ATL21C antibodies in Western blot analysis?

For optimal Western blot results with ATL21C antibodies, researchers should consider:

• Sample preparation: Use plant tissue extraction buffers containing protease inhibitors (e.g., 50mM Tris-HCl, pH 7.5, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, with protease inhibitor cocktail).

• Protein separation: Use 10-12% SDS-PAGE gels to effectively resolve the ATL21C protein.

• Antibody dilution: Start with a 1:1000 dilution of the primary antibody and optimize as needed.

• Validation controls: Include both positive controls (wild-type Arabidopsis extracts) and negative controls (ATL21C knockout mutants if available).

• Expected results: Look for a band at approximately 20-25 kDa based on the predicted molecular weight of the ATL21C protein.

How can immunoprecipitation experiments be optimized for ATL21C protein complexes?

For successful immunoprecipitation of ATL21C and its interaction partners:

• Lysate preparation: Use non-denaturing lysis buffers to preserve protein-protein interactions.

• Pre-clearing: Incubate lysates with Protein A/G beads prior to adding antibodies to reduce non-specific binding.

• Antibody binding: Use 2-5 μg of ATL21C antibody per 500 μg of total protein lysate, incubating overnight at 4°C with gentle rotation.

• Washing conditions: Perform 4-5 washes with buffer containing reduced detergent concentration to remove non-specific interactions while preserving specific complexes.

• Elution strategies: Compare gentle elution (pH shift) versus boiling in SDS buffer, depending on downstream applications.

How can ATL21C antibodies be utilized to study ubiquitination pathways in plants?

ATL21C antibodies enable several sophisticated approaches to investigate plant ubiquitination pathways:

• In vitro ubiquitination assays: Immunopurify ATL21C using the antibody, then combine with E1, E2 enzymes, ubiquitin, and potential substrates to assess E3 ligase activity.

• Substrate identification: Use ATL21C antibodies for immunoprecipitation followed by mass spectrometry to identify proteins targeted by this E3 ligase.

• Ubiquitin chain specificity: Combine ATL21C antibodies with chain-specific ubiquitin antibodies (K48, K63, etc.) to determine the type of ubiquitin linkages formed.

• Stress response studies: Track changes in ATL21C-mediated ubiquitination under various environmental stresses or pathogen challenges.

• Subcellular localization: Perform immunofluorescence studies with ATL21C antibodies to determine where the protein functions within plant cells.

What approaches can be used to integrate ATL21C antibody data with transcriptomics?

Researchers can combine ATL21C antibody-generated data with transcriptomic approaches through:

• Correlation analysis: Compare protein levels detected by ATL21C antibodies with mRNA expression data from RNA-seq or qPCR across different tissues or conditions.

• Chromatin immunoprecipitation (ChIP): If ATL21C affects gene expression directly or indirectly, researchers can correlate ChIP data (using transcription factor antibodies) with ATL21C protein levels.

• Expression validation: Validate RNA-seq findings by confirming protein-level changes using ATL21C antibodies in Western blots or immunohistochemistry.

• Post-transcriptional regulation studies: Investigate discrepancies between mRNA levels and protein abundance to identify potential post-transcriptional or post-translational regulatory mechanisms.

• Time-course studies: Track dynamic changes in both transcript and protein levels following stimuli to understand the temporal relationship between transcription and protein accumulation.

How can researchers address non-specific binding issues with ATL21C antibodies?

When encountering non-specific binding with ATL21C antibodies, consider these methodological solutions:

• Blocking optimization: Test different blocking agents (5% BSA, 5% non-fat milk, commercial blockers) and extended blocking times (2-3 hours).

• Antibody dilution series: Perform a dilution series (1:500 to 1:5000) to identify the optimal concentration that minimizes background while maintaining specific signal.

• Wash buffer optimization: Increase wash stringency by adding more detergent (0.1-0.3% Tween-20) or salt (up to 500 mM NaCl) to wash buffers.

• Antibody pre-absorption: Incubate the antibody with plant extracts from ATL21C knockout lines to remove antibodies that bind to non-specific epitopes.

• Secondary antibody controls: Run controls with only secondary antibody to identify background from this source.

What are the most common pitfalls in quantitative analysis of ATL21C expression?

Researchers should be aware of these challenges when quantifying ATL21C expression:

• Reference protein selection: Choose appropriate loading controls that remain stable under your experimental conditions.

• Dynamic range limitations: Ensure signal intensity falls within the linear range of detection to avoid saturation effects.

• Antibody batch variation: Document lot numbers and perform calibration experiments when switching to new antibody batches.

• Sample preparation inconsistencies: Standardize extraction methods to ensure comparable protein recovery across samples.

• Normalization approaches: Select appropriate normalization strategies based on your experimental design (total protein staining vs. housekeeping proteins).

How does ATL21C research compare to studies on other RING-finger proteins in plants?

Research on ATL21C should be contextualized within the broader RING-finger protein family:

• Evolutionary conservation: Compare ATL21C with other ATL family members in Arabidopsis and across plant species to understand conserved domains and functions.

• Substrate specificity: Investigate whether ATL21C targets unique substrates compared to other RING-finger E3 ligases or shares common targets.

• Regulatory mechanisms: Examine whether ATL21C is regulated through similar post-translational modifications or protein-protein interactions as other family members.

• Functional redundancy: Assess potential functional overlap with other RING-finger proteins through genetic studies and protein localization comparisons.

• Stress responsiveness: Compare the transcriptional and post-translational regulation of ATL21C with other family members under various biotic and abiotic stresses.

What methodological approaches can distinguish between isoforms of ATL21C?

When investigating potential ATL21C isoforms, researchers should consider:

• Isoform-specific antibodies: Develop antibodies targeting unique regions of different ATL21C isoforms.

• Mass spectrometry: Use targeted proteomics approaches to identify and quantify specific peptides unique to each isoform.

• RT-PCR strategies: Design primers spanning exon junctions specific to different splice variants.

• Protein separation techniques: Optimize gel systems to resolve closely related isoforms with small molecular weight differences.

• Genetic complementation: Express individual isoforms in knockout backgrounds to assess their functional equivalence or specificity.

How can ATL21C antibodies contribute to understanding plant stress responses?

ATL21C antibodies can advance plant stress response research through:

• Stress-induced modification tracking: Monitor post-translational modifications of ATL21C under different stress conditions.

• Dynamic protein interaction networks: Identify stress-specific protein interaction partners through co-immunoprecipitation under various stress treatments.

• Temporal expression profiling: Track the timing of ATL21C protein accumulation relative to transcriptional changes during stress responses.

• Cell-type specific expression: Use immunohistochemistry to determine if ATL21C expression is restricted to specific cell types during stress responses.

• Cross-species conservation: Compare ATL21C expression and function across multiple plant species to identify conserved stress response mechanisms.

What emerging technologies might enhance ATL21C antibody-based research?

Researchers should consider integrating these emerging technologies:

• Proximity labeling: Combine ATL21C antibodies with BioID or APEX2 approaches to identify proteins in close proximity to ATL21C in living cells.

• Super-resolution microscopy: Apply techniques like STORM or PALM with ATL21C antibodies to visualize subcellular localization at nanometer resolution.

• Single-cell proteomics: Adapt ATL21C antibody protocols for use in emerging single-cell protein analysis platforms.

• CRISPR-based tagging: Generate endogenously tagged ATL21C to complement antibody-based detection with alternative epitope tags.

• Protein-protein interaction visualization: Implement techniques like BiFC (Bimolecular Fluorescence Complementation) or FRET to visualize ATL21C interactions in living plant cells.

Integrating these advanced approaches with traditional antibody-based techniques will significantly enhance our understanding of ATL21C function in plant biology and potentially reveal new applications for studying ubiquitination pathways in plants.