ICT1 Antibody

What is ICT1 and why is it important in research?

ICT1, also known as 39S ribosomal protein L58 (MRPL58), is an integral component of the mitochondrial ribosome that functions as a peptidyl-tRNA hydrolase. It has several synonyms including Digestion substraction 1 (DS-1), Immature colon carcinoma transcript 1 protein, and Large ribosomal subunit protein mL62 . ICT1 plays a crucial role in ribosome rescue mechanisms and translation termination at non-standard stop codons in mammalian mitochondria . It is particularly significant because it can rescue stalled ribosomes not only at the ends of mRNAs but also in the middle of mRNAs, and even without mRNAs present, making it a versatile player in mitochondrial translation quality control . Research on ICT1 provides valuable insights into mitochondrial translation mechanisms and potentially mitochondrial diseases.

What types of ICT1 antibodies are commercially available?

Researchers have access to both polyclonal and monoclonal antibodies targeting various epitopes of ICT1. Available options include rabbit polyclonal antibodies to DS-1 that react with human, mouse, and rat samples, mouse monoclonal antibodies [PAT1E9A] to DS-1 that specifically target human samples, and rabbit polyclonal antibodies to peptidyl-tRNA hydrolase ICT1 that are human-specific . The diversity of available antibodies allows researchers to select the most appropriate reagent based on their experimental design, target species, and application requirements.

What are the validated applications for ICT1 antibodies?

ICT1 antibodies have been validated for several laboratory techniques:

These applications allow researchers to detect, quantify, and localize ICT1 in various experimental contexts .

How should ICT1 antibodies be used in ribosomal rescue studies?

When studying ICT1's role in ribosomal rescue, researchers should consider the distinction between integrated ICT1 (part of the mitoribosome) and purified/free ICT1. Studies have shown that the codon-independent peptide-release activity on 55S ribosomes is only observed when purified ICT1 is added to a system already containing the 55S-integrated ICT1 . This suggests that the integrated ICT1 lacks peptide-release activity, contrary to previous models.

Methodology for such studies typically involves:

• Isolation of mitochondrial ribosomes (55S)

• Setting up in vitro translation systems with stalled ribosomal complexes

• Adding purified ICT1 to the system

• Measuring peptide release through radioactive labeling (e.g., using f[14C]Met)

• Comparing activity with controls lacking ICT1 or containing mutant versions of ICT1

These approaches allow researchers to quantitatively assess ICT1's rescue activity in different contexts.

What controls should be included when validating ICT1 antibody specificity?

When validating ICT1 antibodies, researchers should implement a multi-faceted approach:

• Positive controls: Include samples known to express ICT1, such as human cell lines with confirmed ICT1 expression

• Negative controls:

  • Primary antibody omission

  • Use of blocking peptides specific to the antibody epitope

  • Samples where ICT1 has been knocked down by siRNA/shRNA

• Cross-reactivity assessment: Test the antibody against recombinant ICT1 and closely related proteins

• Band size verification: Confirm detection at the expected molecular weight (27-28 kDa for human ICT1)

• Subcellular localization confirmation: ICT1 should predominantly show mitochondrial localization

These controls help ensure that observed signals genuinely represent ICT1 and not non-specific binding or cross-reactivity with related proteins.

How can researchers distinguish between integrated and non-integrated ICT1 in experimental systems?

Distinguishing between integrated (mitoribosome-bound) and non-integrated (free) ICT1 is crucial for understanding its dual functions. Researchers can employ several approaches:

• Ribosome fractionation: Using sucrose gradient ultracentrifugation to separate mitochondrial ribosomes from free proteins, followed by Western blot analysis with ICT1 antibodies

• Stoichiometric analysis: Quantifying the ratio of ICT1 to other mitoribosomal proteins to determine the proportion of integrated ICT1

• Functional assays: Comparing peptide-release activity in systems with and without additional purified ICT1

• Immunoprecipitation: Using antibodies against other mitoribosomal proteins to co-precipitate integrated ICT1, leaving free ICT1 in the supernatant

• Structural studies: Using cryo-EM to visualize the positioning of ICT1 within the mitoribosomal structure

Research has shown that purified ICT1 binds stoichiometrically to mitochondrial ribosomes in addition to the integrated copy, suggesting that both forms have distinct roles .

What experimental approaches are recommended for studying ICT1's role in translation termination at non-standard stop codons?

ICT1 plays a role in translation termination at non-standard stop codons (AGA/G) in mammalian mitochondria. To investigate this function, researchers can use:

• In vitro translation systems:

  • E. coli-based reconstituted systems of coupled transcription-translation

  • Mitochondrial translation systems with purified components

  • Systems programmed with mRNAs encoding short polypeptides with different terminal codons

• Comparative analysis with other release factors:

  • Compare ICT1 activity with RF1Lmt/mtRF1a on standard and non-standard stop codons

  • Assess competition or cooperation between factors

• Mutational analysis:

  • Generate ICT1 mutants affecting the unique insertion sequence in the N-terminal domain, which is essential for peptide release

  • Test these mutants in ribosome rescue assays

• Ribosome stalling models:

  • Create different types of stalled ribosomes (e.g., at mRNA 3' ends, in the middle of mRNAs, or non-programmed ribosomes)

  • Compare ICT1's activity across these contexts

Studies have shown that ICT1 can release oligopeptides from stalled ribosomes programmed with stop(UAA) or stall(AGA) mRNAs, though with varying efficiency (approximately 0.3 pmol polypeptides with stop/stall mRNAs versus 2.0 pmol with nonstop mRNAs) .

How should researchers interpret contradictory results when detecting ICT1 with different antibodies?

When faced with contradictory results using different ICT1 antibodies, researchers should consider:

• Epitope differences: Different antibodies target distinct regions of ICT1, which may be differentially accessible depending on:

  • ICT1's integration status in the mitoribosome

  • Post-translational modifications

  • Protein-protein interactions

  • Conformational states

• Antibody specificity: Evaluate each antibody's cross-reactivity profile:

  • Polyclonal antibodies may detect multiple epitopes but risk non-specific binding

  • Monoclonal antibodies offer higher specificity but may miss ICT1 if the epitope is masked

• Experimental conditions: Assess whether differences in protocols could explain discrepancies:

  • Sample preparation methods

  • Denaturing vs. native conditions

  • Buffer compositions

  • Incubation times and temperatures

• Validation approach: Use complementary techniques to resolve contradictions:

  • Combine results from multiple antibodies

  • Employ alternative detection methods (mass spectrometry, RNA interference)

  • Correlate antibody results with functional assays

The contradictory results may actually reveal important biological insights about ICT1's diverse structural states or functions rather than representing technical artifacts.

What factors can affect the reproducibility of experiments using ICT1 antibodies?

Several factors can impact experimental reproducibility when working with ICT1 antibodies:

• Sample preparation variables:

  • Freeze-thaw cycles (although studies suggest ICT1 detection is relatively stable through multiple freeze-thaw cycles)

  • Storage conditions and duration

  • Protein extraction methods

  • Cell or tissue types

• Antibody batch variation:

  • Lot-to-lot differences in antibody production

  • Storage and handling conditions

  • Antibody age and potential degradation

• Technical parameters:

  • Blocking reagents and duration

  • Antibody concentration and incubation times

  • Detection methods and sensitivity

  • Washing stringency

• Biological variables:

  • ICT1 expression levels across different cell types

  • Mitochondrial content variation

  • Cell cycle stage

  • Stress conditions affecting mitochondrial function

To enhance reproducibility, researchers should standardize protocols, maintain detailed records of antibody lots and experimental conditions, and include appropriate controls in each experiment.

How might ICT1 antibodies be used to investigate mitochondrial ribosome quality control mechanisms?

ICT1 antibodies present valuable tools for investigating mitochondrial ribosome quality control through several innovative approaches:

• Spatial and temporal dynamics:

  • Using fluorescently-labeled ICT1 antibodies for live-cell imaging

  • Tracking ICT1 recruitment to stalled ribosomes under various stress conditions

  • Analyzing co-localization with other quality control factors

• Pathological conditions:

  • Examining ICT1 localization and abundance in mitochondrial disease models

  • Assessing changes in ICT1-ribosome interactions during cellular stress

  • Investigating ICT1's role in neurodegenerative disorders with mitochondrial dysfunction

• Integration with structural biology:

  • Using ICT1 antibodies to purify ribosomes at different stages of rescue

  • Combining with cryo-EM to visualize conformational changes during rescue

  • Identifying novel interacting partners through immunoprecipitation followed by mass spectrometry

• Therapeutic opportunities:

  • Developing antibody-based tools to modulate ICT1 activity

  • Using ICT1 antibodies to screen for compounds that affect ribosome rescue

  • Exploring ICT1 as a biomarker for mitochondrial translation defects

These approaches could significantly advance our understanding of how mitochondria maintain translational fidelity and respond to stress conditions .

What are the current knowledge gaps in understanding ICT1 function that new antibodies might help resolve?

Despite significant progress, several knowledge gaps remain in ICT1 research that could be addressed with improved antibodies:

• Structural dynamics:

  • How does the unique insertion sequence in ICT1's N-terminal domain contribute to its function?

  • What conformational changes occur during peptide release?

  • How does ICT1 recognize different ribosomal substrates?

• Regulatory mechanisms:

  • How is ICT1 expression regulated under different cellular conditions?

  • Are there post-translational modifications that affect ICT1 activity?

  • What factors control the balance between integrated and free ICT1?

• Interaction network:

  • What proteins interact with ICT1 during ribosome rescue?

  • How does ICT1 cooperate with or compete against other release factors?

  • What determines substrate specificity for ICT1?

• Pathological relevance:

  • How does ICT1 dysfunction contribute to mitochondrial diseases?

  • Can ICT1 activity be therapeutically modulated?

  • Is ICT1 involved in aging-related mitochondrial decline?

New, highly specific antibodies that can distinguish between different functional states of ICT1 would help researchers address these questions and advance our understanding of mitochondrial translation quality control .