ELAC1 Antibody

What is ELAC1 and what is its primary biological function?

ELAC1 (elaC homolog 1) is a specialized tRNA repair enzyme in vertebrates that specifically removes 2',3'-cyclic phosphates from tRNAs cleaved during ribosome stalling. Unlike its paralog ELAC2 (which functions in tRNA biogenesis), ELAC1 is optimized for tRNA recycling following cleavage by ANKZF1 on stalled ribosomes . The protein functions by recognizing and acting on the ribose of N73 of tRNA substrates, converting various starting products to the exact substrate needed for CCA addition, which makes ELAC1 specifically tailored for tRNA repair . This function is critical for maintaining proper translation and protein quality control mechanisms.

What are the key applications for ELAC1 antibodies in research?

ELAC1 antibodies are primarily utilized in the following research applications:

The antibodies require optimization in each experimental system to achieve optimal results, with sample-dependent considerations that may require titration .

What are the molecular characteristics of ELAC1 protein relevant for antibody detection?

ELAC1 protein has the following characteristics important for antibody-based detection:

These properties are crucial for proper identification and validation when using antibodies against ELAC1 .

How do ELAC1 and ELAC2 differ functionally and why is this distinction important?

ELAC1 and ELAC2 represent a case of evolutionary duplication and specialization in vertebrates. The key differences include:

• Substrate preference: ELAC1 activity drops off sharply with tRNA trailer lengths between 2-5 nucleotides, while ELAC2 efficiently cleaves trailers of all lengths examined

• Biological role: ELAC1 is optimized for tRNA recycling, whereas ELAC2 is the essential RNase Z isoform required for removing 3′ trailers during tRNA biogenesis

• Evolutionary significance: The duplication allows specialized handling of tRNA processing (ELAC2) and repair (ELAC1)

Understanding this distinction is critical when designing experiments to study specific aspects of tRNA metabolism, as targeting one without affecting the other requires careful experimental design.

What experimental protocols yield optimal results when using ELAC1 antibodies for Western blotting?

For optimal Western blot results with ELAC1 antibodies, researchers should consider the following protocol elements:

• Sample preparation: Mouse liver tissue has shown consistent positive detection

• Antibody dilution: Begin with 1:1000 dilution and optimize between 1:1000-1:6000 range based on signal intensity

• Buffer composition: Use PBS with 0.02% sodium azide and 50% glycerol pH 7.3 for antibody storage and dilution

• Controls: Include wild-type and ELAC1 knockout samples when possible to validate specificity

• Detection method: Both chemiluminescence and fluorescence-based methods are suitable

The molecular weight of 45-48 kDa should be monitored as the expected band size for ELAC1 .

What methodological considerations should be made when using ELAC1 antibodies for immunohistochemistry?

When performing immunohistochemistry with ELAC1 antibodies, consider these methodological approaches:

• Antigen retrieval: Use TE buffer pH 9.0 for optimal results; citrate buffer pH 6.0 may be used as an alternative

• Antibody dilution: Start with 1:200 and optimize within the 1:50-1:500 range

• Incubation conditions: Overnight incubation at 4°C generally yields better results than shorter incubations

• Detection system: Rabbit IgG-compatible detection systems are required as ELAC1 antibodies are typically rabbit polyclonal

• Positive control: Human liver cancer tissue has been confirmed as a reliable positive control

Researchers should always perform titration experiments to determine optimal conditions for their specific tissue samples and fixation methods.

How can researchers validate ELAC1 antibody specificity in their experimental systems?

Validation of ELAC1 antibody specificity requires multiple complementary approaches:

• Genetic validation: Compare staining patterns between wild-type and ELAC1 knockout samples

• Molecular weight verification: Confirm that the detected band matches the expected 45-48 kDa size

• Peptide competition: Pre-incubate antibody with the immunizing peptide to demonstrate signal reduction

• Multiple antibody comparison: Use antibodies from different vendors or those recognizing different epitopes

• Orthogonal methods: Confirm ELAC1 expression using RNA detection methods (qPCR, RNA-seq)

• Functional validation: Demonstrate that phenotypes observed upon ELAC1 depletion are consistent with its known role in tRNA repair

This multi-faceted validation approach ensures that experimental observations genuinely reflect ELAC1 biology rather than antibody artifacts.

How can ELAC1 antibodies be used to investigate tRNA recycling dynamics during cellular stress?

ELAC1 antibodies can elucidate tRNA recycling dynamics through these advanced approaches:

• Co-immunoprecipitation: Use ELAC1 antibodies to pull down protein complexes involved in tRNA recycling

• Proximity labeling: Combine with BioID or APEX approaches to identify proteins in the vicinity of ELAC1 during stress

• Stress induction experiments: Monitor ELAC1 localization and association with tRNAs during translation inhibition (e.g., cycloheximide treatment, which induces ribosome stalling)

• Pulse-chase studies: Track newly synthesized ELAC1 during stress recovery to assess turnover rates

• Imaging approaches: Combine with fluorescent tRNA probes to visualize co-localization during stress

Research has demonstrated that cells lacking ELAC1 specifically accumulate unrepaired tRNA intermediates upon induction of ribosome stalling with cycloheximide, providing a clear readout for ELAC1 function .

What experimental approaches can identify ELAC1-specific contributions to tRNA repair versus those of ELAC2?

To distinguish ELAC1-specific contributions from ELAC2, consider these methodological approaches:

• Substrate specificity assays: Utilize tRNAs with different 3' trailer lengths (ELAC1 activity drops sharply with trailers of 2-5 nucleotides, while ELAC2 cleaves all lengths)

• Selective knockdown: Compare phenotypes of ELAC1 versus ELAC2 knockout/knockdown

• Rescue experiments: Test whether ELAC1 or ELAC2 can rescue defects in cells lacking the other protein

• In vitro activity assays: Compare enzymatic activity using purified proteins on various tRNA substrates

• Structure-function analysis: Use mutational analysis targeting the active site (e.g., H64A mutation abolishes ELAC1 repair activity)

Research has shown that lysates lacking ELAC1 were not impaired in processing ΔCCA-trailer, consistent with ELAC1 not being a major contributor to tRNA biogenesis, which is primarily handled by ELAC2 .

How can researchers quantitatively assess ELAC1's 2',3'-cyclic phosphate removal activity?

For quantitative assessment of ELAC1's activity, these methodological approaches are recommended:

• Radiolabeling assays: Using [α-32P]CTP incorporation to measure repaired tRNAs that can accept CCA addition

• Gel electrophoresis analysis: Monitoring the conversion of ΔCCA tRNA to full-length tRNA with intact CCA end

• Biochemical fractionation: Using column chromatography to isolate and quantify ELAC1 activity

• Recombinant protein assays: Comparing wild-type ELAC1 with inactive mutants (H64A) in repairing 2',3'-cyclic phosphates

• Coupled enzyme assays: With TRNT1 (CCA-adding enzyme) to measure the complete repair and CCA addition process

The research shows that ELAC1 efficiently repairs ΔCCA-HDV for CCA addition by TRNT1, with activity comparable to T4 PNK, while a single H64A mutation disrupts the active site and abolishes repair activity .

What are the key considerations when analyzing ELAC1 knockdown or knockout phenotypes?

When analyzing ELAC1 knockout phenotypes, researchers should consider:

• tRNA recycling intermediates: Measure accumulation of unrepaired tRNAs by incubating RNA with radiolabeled CTP and TRNT1

• Stress responses: Examine outcomes during normal conditions versus ribosome stalling (e.g., cycloheximide treatment)

• Compensatory mechanisms: Assess whether ELAC2 or other enzymes compensate for ELAC1 loss

• Tissue-specific effects: Consider variable expression patterns of ELAC1 across tissues (e.g., liver shows strong expression)

• Temporal dynamics: Acute versus chronic loss may reveal different phenotypes due to adaptation

Research has demonstrated that ELAC1 knockout cells specifically accumulate unrepaired tRNAs upon induction of ribosome stalling, providing a clear cellular phenotype that can be measured .

How should researchers address inconsistent Western blot results with ELAC1 antibodies?

When troubleshooting inconsistent Western blot results:

• Optimize protein extraction: ELAC1 requires specific extraction conditions; test different lysis buffers

• Modify blocking conditions: Test different blocking agents (BSA vs. milk) and concentrations

• Adjust antibody incubation: Try longer incubation times (overnight at 4°C) or different dilutions (1:1000-1:6000)

• Sample preparation: Freshly prepared lysates generally yield better results than frozen-thawed samples

• Positive control inclusion: Always include mouse liver tissue as a positive control for ELAC1 detection

• Storage conditions: Ensure antibody is stored at -20°C, and aliquoting is unnecessary for -20°C storage

The observed molecular weight of 45-48 kDa should be used as reference for proper band identification .

What methodological approaches can resolve contradictory findings between ELAC1 antibody-based detection and functional assays?

To resolve contradictions between antibody detection and functional results:

• Epitope mapping: Determine if post-translational modifications might mask epitopes

• Multiple antibody validation: Use antibodies recognizing different epitopes of ELAC1

• Activity-based probes: Develop probes that bind to active ELAC1 to distinguish functional protein

• Subcellular fractionation: Assess whether ELAC1 is present but sequestered in unexpected compartments

• Protein complex analysis: Determine if ELAC1 functions within protein complexes that might conceal epitopes

• RNA-binding state: Consider whether RNA binding affects antibody recognition

Research has shown that ELAC1 is necessary and sufficient to remove 2',3'>p from ΔCCA tRNAs for recycling, providing a functional readout that can be compared with antibody detection results .

How can biophysics-informed computational models enhance ELAC1 antibody specificity analysis?

Computational approaches for antibody specificity analysis include:

• Binding mode identification: Computational models can identify different binding modes associated with particular ligands

• Specificity profile customization: Models trained on experimental data can predict and generate specific variants beyond those observed experimentally

• Cross-reactivity prediction: Identify potential off-target binding by analyzing structural similarities

• Epitope mapping: Predict antigenic regions likely to elicit specific antibody responses

• Molecular dynamics simulations: Assess conformational changes that might affect epitope accessibility

Research demonstrates that biophysics-informed models can disentangle multiple binding modes associated with specific ligands, enabling prediction and generation of antibody variants not present in initial libraries with customized specificity profiles .

What emerging methodologies might enhance ELAC1 antibody-based research in the coming years?

Emerging methodologies that may advance ELAC1 research include:

• Single-molecule imaging: Tracking individual ELAC1 molecules during tRNA repair processes

• CRISPR-based tagging: Endogenous tagging for live-cell imaging of ELAC1 dynamics

• Mass spectrometry-based interactomics: Comprehensive mapping of ELAC1 interaction partners

• Nanobody development: Smaller antibody formats for improved access to restricted epitopes

• Synthetic antibody libraries: Custom-designed antibodies with enhanced specificity for ELAC1

These approaches could provide unprecedented insights into ELAC1 function, particularly its dynamic behavior during cellular stress responses and ribosome stalling events.

How might ELAC1 antibodies contribute to understanding the cross-talk between tRNA repair and other cellular processes?

ELAC1 antibodies can illuminate connections between tRNA repair and broader cellular processes through:

• Proximity-dependent biotinylation: Identify proteins physically near ELAC1 during different cellular states

• Phospho-proteomics: Assess how ELAC1 function changes with post-translational modifications

• Co-localization studies: Examine ELAC1 relationships with stress granules, P-bodies, and ribosomes

• Multi-omics integration: Combine proteomics and transcriptomics to map ELAC1 regulatory networks

• Temporal analysis: Track ELAC1 localization and interactions throughout the cell cycle

Understanding these relationships may reveal new roles for ELAC1 beyond tRNA repair and provide insights into cellular stress response integration.