ABCE1 Antibody

What is ABCE1 and why is it significant for research?

ABCE1 (ATP-binding cassette, sub-family E, member 1) is a highly conserved protein present in eukaryotes and archaea that is essential for cell viability. Originally identified as an RNase L inhibitor, ABCE1 is now recognized as a crucial translation factor involved in multiple stages of eukaryotic translation and ribosome biogenesis . Its significance stems from its diverse functional roles, including:

• Ribosome recycling through twin-ATPase activity

• Critical involvement in S phase progression and DNA replication

• Regulation of histone biosynthesis

• Association with HIV capsid assembly and endogenous suppression of RNA interference

• Implication in cancer pathogenesis through its roles in cell proliferation

Research on ABCE1 continues to expand our understanding of fundamental cellular processes and potential therapeutic targets.

What are the recommended applications and dilutions for ABCE1 antibody?

ABCE1 antibodies such as 28548-1-AP have been validated for multiple applications with specific recommended dilutions for optimal results:

It is crucial to note that optimal dilutions are sample-dependent, and researchers should perform titration experiments in their specific testing systems to achieve optimal results .

What cell lines and tissues show positive reactivity with ABCE1 antibody?

ABCE1 antibody has demonstrated positive reactivity in multiple cell types and tissues:

For IHC applications, antigen retrieval is suggested with TE buffer pH 9.0, though citrate buffer pH 6.0 may serve as an alternative .

How can researchers effectively detect changes in ABCE1 expression during cell cycle progression?

For investigating ABCE1 expression throughout the cell cycle, researchers should consider:

• Synchronization protocols: Double thymidine block effectively synchronizes cells at the G1/S boundary for S phase analysis. Studies show ABCE1 protein expression remains stable throughout S phase despite its critical role in this phase .

• Controls for validation: Include S phase-specific markers such as SLBP (stem-loop binding protein), which demonstrates expected accumulation during S phase while ABCE1 remains relatively constant .

• Detection methods: Combine flow cytometry for cell cycle analysis with protein detection methods. Research shows that ABCE1 depletion results in a 1.7-fold increase of cells in S phase and a 0.6-fold decrease in G0/G1 phase, detectable by day 4 post-transfection .

• Time-course design: Monitor expression from 0-8 hours post-synchronization release to capture the entire S phase progression .

ABCE1 exhibits non-periodic expression despite its critical role in S phase, suggesting its function is regulated through mechanisms other than expression level fluctuations.

What approaches can distinguish between ABCE1's roles in general translation versus specific S phase functions?

To differentiate between ABCE1's general translation functions and its specific role in S phase progression:

• Timing analysis: Perform parallel studies on translation efficiency and cell cycle progression. Research demonstrates that S phase accumulation occurs before significant reduction in general translation (while translation remains at 60-80% of control values), suggesting independent regulatory mechanisms .

• Specific target analysis: Monitor synthesis of S phase-specific proteins, particularly histones. ABCE1 depletion significantly reduces both histone mRNA and protein levels .

• BrdU incorporation assays: Following synchronization and release into S phase, use BrdU labeling to assess DNA synthesis rates. In ABCE1-depleted cells, the proportion of DNA-synthesizing cells is significantly lower at 2 and 4 hours after release compared to control .

• Translation specificity assays: Examine whether ABCE1 depletion affects all translated proteins equally or shows preference for specific transcripts through metabolic labeling and proteomics approaches.

This multi-faceted approach helps distinguish between general translation defects and specific impacts on S phase-related functions.

What methodologies best capture ABCE1's conformational dynamics during ribosome recycling?

Recent research has revealed that ABCE1 exists in a dynamic equilibrium across three distinct conformational states (open, intermediate, and closed), challenging previous static two-state models . To effectively study these dynamics:

• Single-molecule FRET: This technique allows direct observation of conformational changes in real-time. It revealed that ABCE1's two ATP sites operate in an asymmetrical fashion across multiple conformational states .

• Integrated biophysical approach: Combine multiple techniques including:

  • FRET for conformational dynamics

  • Ribosome association assays

  • ATP hydrolysis measurements

  • Structural analysis

• Time-resolved measurements: Track the transitions between states over time to understand the kinetics and regulatory mechanisms underlying ABCE1 function.

• Ribosome interaction studies: Examine how ribosome binding influences the conformational landscape of ABCE1, as research indicates ribosome interaction creates asymmetric effects on the two ATP sites .

This dynamic-based approach represents a paradigm shift from structure-based deterministic models to better understand the mechanochemical coupling in ABC proteins.

What are common issues when using ABCE1 antibody in Western blot and how can they be resolved?

Follow the specific Western blot protocol provided with ABCE1 antibody, ensuring proper sample preparation and loading appropriate positive controls.

How should researchers interpret cell cycle data following ABCE1 depletion?

When analyzing cell cycle data after ABCE1 knockdown:

• Expected pattern: Look for accumulation of cells in S phase (approximately 1.7-fold increase) with corresponding decrease in G0/G1 phase (approximately 0.6-fold) .

• Timing considerations: Cell cycle effects typically appear by day 4 post-transfection and persist through day 6, preceding dramatic defects in general translation .

• Cell type variations: While the S phase accumulation pattern is consistent across cell types (observed in both HEK293 and HeLa cells), the magnitude may vary .

• Synchronization analysis: After synchronization, expect normal G1/S transition but delayed progression through mid-S phase (between 2-4 hours after release) .

• DNA synthesis correlation: Correlate cell cycle data with BrdU incorporation results, expecting reduced DNA synthesis coinciding with S phase accumulation .

These interpretations help distinguish between direct effects on DNA replication machinery versus secondary effects from translation impairment.

How can ABCE1 antibody be used to investigate the relationship between translation and cancer?

ABCE1 antibodies serve as valuable tools for investigating the link between translation dysregulation and cancer development:

• Expression analysis: Multiple studies report upregulated ABCE1 expression in various cancers. IHC with ABCE1 antibody (dilution 1:50-1:500) can be used to assess expression levels in tumor tissues compared to normal tissues .

• Functional studies: Combining ABCE1 antibody detection with siRNA knockdown approaches reveals that inhibition of ABCE1 can efficiently suppress tumor cell proliferation .

• Pathway analysis: ABCE1's connection to RNase L, implicated in hereditary prostate cancer, can be investigated through co-immunoprecipitation and expression correlation studies .

• Therapeutic target validation: Following potential anti-ABCE1 treatments, antibody-based detection can confirm target engagement and downstream effects on cancer pathways.

When interpreting results, consider that tumor cells at low confluence may show stronger phenotypic responses to ABCE1 depletion, suggesting density-dependent effects that could influence therapeutic targeting strategies .

What experimental design best addresses ABCE1's dual role in DNA replication and histone synthesis?

To investigate the interconnected roles of ABCE1 in DNA replication and histone synthesis:

• Sequential analysis approach:

  • First establish ABCE1 depletion using siRNA (70-90% knockdown at protein level is optimal)

  • Monitor cell cycle progression by flow cytometry

  • Assess DNA synthesis through BrdU incorporation

  • Measure histone mRNA and protein levels

  • Evaluate general translation efficiency

• Time-resolved experiments: Studies show that DNA synthesis inhibition occurs at 2-4 hours after release into S phase in ABCE1-depleted cells, coinciding with the period when S phase progression is delayed .

• Mechanistic dissection:

  • Use S phase checkpoint inhibitors to determine if checkpoint activation mediates the observed phenotypes

  • Introduce exogenous histones to test if histone deficiency is the primary cause of replication defects

  • Employ DNA damage markers to assess if replication stress contributes to S phase delay

This comprehensive approach helps establish whether ABCE1's primary function in S phase is related to histone synthesis regulation, with DNA replication defects occurring as a secondary consequence.

How might studying ABCE1 conformational dynamics lead to new therapeutic approaches?

The recent discovery that ABCE1 operates across three distinct conformational states rather than two opens novel research avenues :

• Structure-function studies: By understanding the precise conformational changes associated with different ABCE1 functions, researchers could design modulators that selectively influence specific activities.

• Cancer therapy approaches: Since ABCE1 is essential for cell proliferation and often upregulated in cancer, compounds that lock ABCE1 in non-functional conformational states could serve as targeted therapies .

• Viral inhibition strategies: Given ABCE1's involvement in HIV capsid assembly, conformational targeting could potentially disrupt viral replication without affecting essential cellular functions .

• Ribosome-targeted therapeutics: The asymmetric influence of ribosome interaction on ABCE1 conformational dynamics suggests possible allosteric regulation points for translation-targeting drugs .

Future research should focus on developing probes that can detect ABCE1 conformational states in living cells and identify small molecules that stabilize specific conformations for therapeutic benefit.

What are the best approaches for investigating tissue-specific functions of ABCE1?

While ABCE1 is universally essential, its roles may vary across tissues:

• Tissue microarray analysis: Use ABCE1 antibody for IHC on tissue microarrays to establish expression patterns across normal and pathological tissues. Current data shows positive reactivity in breast cancer, ovary tumor, and gliomas tissues .

• Conditional knockout models: Generate tissue-specific or inducible ABCE1 knockout models to overcome the embryonic lethality of complete knockout.

• Ex vivo tissue culture: Combine siRNA approaches with organoid or tissue slice cultures to study tissue-specific phenotypes while maintaining proper cellular architecture.

• Multi-omics integration: Correlate ABCE1 expression with tissue-specific transcriptomes and proteomes to identify potential tissue-specific interaction partners and regulated transcripts.

These approaches will help distinguish universal functions of ABCE1 from tissue-specific roles, potentially revealing new therapeutic opportunities with reduced side effects.