ABCE1 Antibody

What is ABCE1 and what are its primary biological functions?

ABCE1 (ATP-binding cassette, sub-family E member 1) is a multifunctional nucleoside-triphosphatase (NTPase) involved in several critical cellular processes. It functions primarily as a ribosome recycling factor, mediating ribosome disassembly by splitting ribosomes into free 60S subunits and tRNA/mRNA-bound 40S subunits . ABCE1 plays essential roles in translation termination through interaction with release factors (like ETF1/eRF1) and in the No-Go Decay (NGD) pathway through interactions with the Pelota-HBS1L complex . Additionally, ABCE1 functions as a negative regulator of the antiviral 2-5A/RNASEL pathway, acting as an RNase L inhibitor . Recent research has revealed its involvement in quality control of mitochondrial outer membrane-localized mRNA translation and PINK1-regulated mitophagy .

What types of ABCE1 antibodies are available for research applications?

Several types of ABCE1 antibodies are available for research, differing in host species, clonality, and conjugation:

Researchers should select antibodies based on their specific application requirements and target species .

What is the molecular weight and structure of the ABCE1 protein?

ABCE1 has a calculated molecular weight of 67 kDa (599 amino acids) and is typically observed at approximately 68 kDa in Western blot analyses . The protein contains two nucleotide-binding domains (NBDs) with ATPase activity, capable of hydrolyzing ATP, GTP, UTP, and CTP . ABCE1's GenBank accession number is BC016283, and its UniProt ID is P61221 . The protein's structure is highly conserved across species, reflecting its essential cellular functions .

What are the recommended protocols for using ABCE1 antibodies in Western blot applications?

For Western blot applications using ABCE1 antibodies, follow these methodological guidelines:

• Sample preparation: ABCE1 has been successfully detected in various cell lines including A549, HEK-293, HeLa, K-562, MCF-7, and NCI-H1299 cells .

• Dilution ratio: The recommended dilution range is typically 1:500-1:5000, with specific antibodies like 28548-1-AP optimally used at 1:500-1:1000 .

• Expected band size: The observed molecular weight is approximately 68 kDa .

• Protocol specifics:

  • Use standard SDS-PAGE separation techniques

  • Transfer to appropriate membrane

  • Block with recommended blocking buffer

  • Incubate with primary antibody at the recommended dilution

  • Follow with appropriate secondary antibody detection system

  • Visualize using chemiluminescence or other detection methods

It is recommended to titrate the antibody concentration in each testing system to obtain optimal results .

How should ABCE1 antibodies be used for immunohistochemistry applications?

For immunohistochemistry (IHC) applications with ABCE1 antibodies, follow these methodological considerations:

• Tissue types: ABCE1 antibodies have been validated in human breast cancer tissue, human ovary tumor tissue, and human gliomas tissue .

• Antigen retrieval: Use TE buffer pH 9.0 for optimal results. Alternatively, citrate buffer pH 6.0 can be used for antigen retrieval .

• Dilution ratio: For IHC applications, the recommended dilution range is 1:50-1:500, with specific optimization required for individual experimental systems .

• Detection methods: Standard immunohistochemistry protocols with appropriate secondary detection systems can be employed .

• Controls: Include appropriate positive and negative controls to validate staining specificity .

The specific protocols should be optimized for each testing system to obtain optimal results .

What are the recommended procedures for immunofluorescence and flow cytometry applications?

For immunofluorescence (IF) and flow cytometry applications using ABCE1 antibodies:

Immunofluorescence (IF/ICC):

• Cell types: Successfully tested in HeLa cells .

• Dilution: Use at 1:50-1:500 dilution .

• Procedure: Follow standard IF protocols with appropriate secondary antibodies or use directly conjugated antibodies like CL594-67960 (excitation/emission maxima: 588 nm/604 nm) .

Flow Cytometry (Intracellular):

• Cell preparation: Successfully validated in HeLa cells .

• Antibody amount: Use 0.40 μg per 10^6 cells in a 100 μl suspension .

• Controls: Include appropriate isotype controls (e.g., Mouse IgG2b for CL594-67960) .

• Cell fixation/permeabilization: Required for intracellular staining of ABCE1 .

For both applications, it is essential to optimize conditions based on specific experimental requirements and follow detailed protocols provided by the manufacturer .

How can ABCE1 antibodies be used to study ribosome recycling and translation termination?

ABCE1 plays a critical role in ribosome recycling by dissociating ribosomes into large and small subunits after translation termination or recognition of stalled ribosomes . Researchers investigating these processes can employ several advanced methodological approaches:

• Co-immunoprecipitation (Co-IP) assays: ABCE1 antibodies can be used to study interactions with translation termination factors (eRF1, eRF3) and ribosomal surveillance factors (Pelota, HBS1L) . These interactions are crucial for understanding how ABCE1 functions in both canonical translation termination and ribosome-associated quality control pathways.

• Subcellular localization studies: Fluorescently conjugated antibodies (such as CL594-67960) can be used to visualize the localization of ABCE1 in relation to ribosomes and translation factors during active translation and stress conditions .

• Polysome profiling: Combined with Western blot analysis using ABCE1 antibodies, this approach can reveal the association of ABCE1 with various ribosomal fractions during translation termination and recycling .

• Ribosome-binding assays: ABCE1 antibodies can be employed to detect and quantify ABCE1 association with ribosomes under different conditions, including the presence of translation inhibitors or stress inducers .

These approaches can provide insights into how ABCE1 functions in normal translation termination and in specialized pathways such as No-Go Decay (NGD) .

What is the relationship between ABCE1 and RNase L, and how can this be studied?

ABCE1 has a complex relationship with RNase L, functioning not only as an inhibitor but potentially also as a cofactor in certain contexts . To investigate this relationship, researchers can use these methodological approaches:

• Direct interaction studies: Co-immunoprecipitation assays have demonstrated that ABCE1 directly interacts with RNase L and Pelota . Researchers can use ABCE1 antibodies to pull down protein complexes and analyze the interaction partners under different cellular conditions.

• Functional assays: The effect of ABCE1 on RNase L activity can be studied using exogenous RNA decay assays, where the half-life of an in vitro transcribed RNA (such as viral RNA) is measured in the presence or absence of ABCE1 .

• Structural analysis: Immunoprecipitated ABCE1-RNase L complexes can be analyzed to determine the binding domains and structural changes that occur during interaction .

• 2-5A pathway studies: ABCE1 has been shown to antagonize the binding of 2-5A (5'-phosphorylated 2',5'-linked oligoadenylates) to RNase L . The effect of ABCE1 on this pathway can be studied using antibodies to detect changes in RNase L dimerization and activation.

Research has shown that in deoxycholate-solubilized cell extracts, RNase L forms dimers even in the absence of 2-5A, and under these conditions, interactions among Pelota, ABCE1, and RNase L have been validated .

How can ABCE1 antibodies be used to study viral infections and host defense mechanisms?

ABCE1 has multiple roles in viral infections and host defense mechanisms. Researchers can employ these methodological approaches:

• Viral infection models: ABCE1 antibodies can be used to monitor changes in ABCE1 expression and localization during viral infections, particularly for HIV-1 and encephalomyocarditis virus (EMCV) .

• HIV-1 capsid assembly studies: ABCE1 may act as a chaperone for post-translational events during HIV-1 capsid assembly . Co-localization studies using fluorescently labeled ABCE1 antibodies can help visualize this process.

• Interferon response pathways: The role of ABCE1 in down-regulating the 2-5A/RNASEL pathway during viral infections can be studied using antibodies to track protein expression and interactions in interferon-stimulated cells .

• RNA decay assays: ABCE1's role in exogenous RNA decay during viral infections can be assessed using methods that measure the half-life of viral RNA in cells with normal or altered ABCE1 expression .

• Knockout/knockdown validation: The specificity of observed effects can be confirmed using ABCE1 knockdown or knockout cells, with antibodies used to validate the reduction in protein levels .

These approaches can help elucidate ABCE1's dual roles in both promoting viral replication (through inhibition of RNase L) and facilitating host defense mechanisms (through exogenous RNA decay) .

What are the optimal storage conditions for ABCE1 antibodies?

Proper storage is crucial for maintaining antibody activity. For ABCE1 antibodies, the following storage conditions are recommended:

• Temperature: Store at -20°C for long-term storage .

• Formulation: Most ABCE1 antibodies are supplied in liquid form with stabilizers:

  • 28548-1-AP: PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • 107804: PBS with 0.1% sodium azide and 50% glycerol, pH 7.3

  • CL594-67960: PBS with 50% Glycerol, 0.05% Proclin300, 0.5% BSA, pH 7.3

• Aliquoting: For most antibodies, aliquoting is unnecessary for -20°C storage, though some preparations (20μl sizes of 28548-1-AP) contain 0.1% BSA .

• Stability: Most ABCE1 antibodies are stable for one year after shipment when stored properly .

• Special considerations for conjugated antibodies: For fluorophore-conjugated antibodies like CL594-67960, avoid exposure to light .

Following these storage recommendations will help maintain antibody performance and specificity over time .

What are common troubleshooting issues when using ABCE1 antibodies?

When working with ABCE1 antibodies, researchers may encounter several technical challenges:

• High background in immunostaining:

  • Solution: Optimize blocking conditions and antibody dilution

  • Use recommended dilution ranges (1:50-1:500 for IHC, 1:500-1:5000 for WB)

  • Consider alternative antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Multiple bands in Western blot:

  • Expected molecular weight: 68 kDa

  • Solution: Optimize sample preparation, ensure complete denaturation

  • Increase washing steps and duration

  • Validate specificity using knockdown/knockout controls

• Weak or no signal:

  • Solution: Titrate antibody concentration for each experimental system

  • Increase antibody incubation time or protein loading

  • Check sample preparation methods to ensure protein integrity

  • Verify expression in your specific cell type or tissue

• Specificity concerns:

  • Multiple publications have validated antibody specificity using knockdown approaches

  • Consider using positive control samples (e.g., A549, HEK-293, HeLa, K-562, MCF-7, NCI-H1299 cells)

• Cross-reactivity issues:

  • Verify species reactivity before use (human, mouse, rat)

  • For untested species, check sequence homology or perform preliminary validation experiments

Carefully optimizing experimental conditions for each specific application and cell/tissue type is essential for obtaining reliable results .

What are the latest discoveries about ABCE1's role in mitochondrial quality control?

Recent research has revealed a previously unknown role for ABCE1 in mitochondrial quality control and mitophagy:

• PINK1-regulated signaling: ABCE1 has been identified as part of the PINK1-regulated signaling pathway involved in mitophagy. Upon mitochondrial damage, ABCE1 is ubiquitinated by CNOT4, generating polyubiquitin signals that recruit autophagy receptors to the mitochondrial outer membrane to initiate mitophagy .

• Quality control of translation: ABCE1 plays a role in quality control of translation specifically for mitochondrial outer membrane-localized mRNAs . This suggests a specialized function in maintaining mitochondrial proteostasis.

• Integration with stress responses: These findings connect ABCE1's canonical role in translation termination and ribosome recycling with mitochondrial homeostasis and cellular stress responses, suggesting a more integrated cellular function than previously recognized .

These discoveries open new research directions for investigating ABCE1's role in mitochondrial diseases and stress-related pathologies .

How is ABCE1 involved in RNA surveillance pathways beyond its inhibitory role on RNase L?

Recent studies have revealed that ABCE1's role in RNA surveillance is more complex than previously thought:

• Positive regulator of exogenous RNA decay: Contrary to its established role as an RNase L inhibitor, research has shown that ABCE1 can act as a positive regulator of exogenous RNA decay . This suggests a context-dependent function in RNA surveillance pathways.

• Complex formation with RNase L and Pelota: ABCE1 directly interacts with both RNase L and Pelota, forming a complex that may facilitate the decay of exogenous RNAs such as viral RNAs . This interaction has been validated through coimmunoprecipitation assays.

• Translation-dependent RNA decay: ABCE1's role in RNA decay appears to be dependent on translation, connecting its canonical function in ribosome recycling with RNA surveillance mechanisms .

• No-Go Decay pathway involvement: ABCE1 is recruited to stalled ribosomes by the Pelota-HBS1L complex and drives the disassembly of these ribosomes, followed by degradation of damaged mRNAs as part of the No-Go Decay (NGD) pathway .

These findings suggest that ABCE1 has evolved to function at the intersection of translation, RNA quality control, and antiviral defense, highlighting its central role in cellular RNA metabolism .

What emerging techniques are being developed for studying ABCE1 function?

Several emerging techniques show promise for advancing our understanding of ABCE1 function:

• CRISPR-based screening approaches: These can identify novel interaction partners and functional pathways involving ABCE1 in various cellular contexts .

• Proximity labeling techniques: BioID or APEX2-based approaches can identify proteins in close proximity to ABCE1 in living cells, providing insights into its dynamic interactome during different cellular processes .

• Single-molecule tracking: This can reveal the dynamics of ABCE1's association with ribosomes during translation termination and recycling in real-time .

• Cryo-electron microscopy: High-resolution structural studies can elucidate the precise molecular mechanisms by which ABCE1 disassembles ribosomes and interacts with other factors like RNase L .

• RNA-protein interaction mapping: Techniques like CLIP-seq can identify the RNA targets that ABCE1 may interact with directly or indirectly, potentially revealing new roles in RNA metabolism .

• Tissue-specific conditional knockout models: These can help delineate the tissue-specific functions of ABCE1 in complex organisms and disease models .

These advanced methodologies, combined with established techniques using validated ABCE1 antibodies, will be crucial for unraveling the multifaceted functions of this essential protein in cellular homeostasis and disease states .

What positive and negative controls should be included when using ABCE1 antibodies?

For rigorous experimental design with ABCE1 antibodies, include these controls:

Positive controls:

• Cell lines with known ABCE1 expression: A549, HEK-293, HeLa, K-562, MCF-7, and NCI-H1299 cells have been validated for ABCE1 detection by Western blot .

• Tissue samples: Human breast cancer tissue, human ovary tumor tissue, and human gliomas tissue have shown positive staining in IHC applications .

• Recombinant ABCE1 protein: Can serve as a positive control in Western blot applications.

Negative controls:

• ABCE1 knockdown/knockout samples: Several publications have validated antibody specificity using KD/KO approaches .

• Isotype controls: For flow cytometry applications, use appropriate isotype controls (e.g., Mouse IgG2b for CL594-67960) .

• Secondary antibody-only controls: To assess background staining in immunohistochemistry and immunofluorescence applications.

• Blocking peptide controls: When available, pre-incubation of the antibody with its immunizing peptide can confirm specificity.

These controls help ensure experimental validity and reliable interpretation of results across different applications .

What factors should be considered when selecting the appropriate ABCE1 antibody for specific research applications?

When selecting an ABCE1 antibody for your research, consider these key factors:

• Application compatibility:

  • For Western blot: 28548-1-AP, 107804, ab250835 are suitable

  • For IHC: 28548-1-AP, 107804, ab250835 are validated

  • For IF/ICC: CL594-67960, ab250835 are recommended

  • For Flow cytometry: CL594-67960, ab250835 are appropriate

  • For IP: ab250835 has been validated

• Species reactivity:

  • Human samples: All listed antibodies work

  • Mouse samples: 28548-1-AP, 107804, ab250835, CL594-67960 are reactive

  • Rat samples: 107804, ab250835, CL594-67960 have been validated

• Clonality considerations:

  • Monoclonal antibodies (ab250835, CL594-67960) offer higher specificity and batch-to-batch consistency

  • Polyclonal antibodies (28548-1-AP, 107804) may provide stronger signals by recognizing multiple epitopes

• Conjugation needs:

  • For direct detection: CL594-67960 (CoraLite®594 conjugated) is suitable for fluorescence applications

  • For conjugation-ready formats: ab250835 (BSA and Azide free) is designed for custom labeling

  • For conventional applications: Unconjugated antibodies (28548-1-AP, 107804) work with standard secondary detection systems

• Experimental conditions:

  • Consider buffer compatibility, fixation requirements, and antigen retrieval methods

  • For IHC applications, note that TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative

Selecting the appropriate antibody based on these factors will help ensure experimental success and reliable results .

How can researchers validate ABCE1 antibody specificity for their experimental system?

To validate ABCE1 antibody specificity in your experimental system:

• Genetic validation approaches:

  • Perform siRNA/shRNA knockdown of ABCE1 and confirm signal reduction

  • Use CRISPR/Cas9-mediated knockout cells as negative controls

  • Overexpress tagged ABCE1 and confirm co-localization with antibody staining

• Biochemical validations:

  • Perform Western blot to confirm single band at expected molecular weight (68 kDa)

  • Use multiple antibodies targeting different epitopes of ABCE1 and compare staining patterns

  • For applications like IP, validate pull-down using Western blot with a different ABCE1 antibody

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide/protein and demonstrate signal reduction

  • This confirms epitope-specific binding

• Cross-species validation:

  • Test antibody reactivity in species with high ABCE1 homology

  • Compare staining patterns across phylogenetically diverse species to assess conservation

• Positive control tissues/cells:

  • Compare staining in tissues/cells with known ABCE1 expression levels (e.g., A549, HEK-293, HeLa cells)

  • Include tissues with variable expression to confirm sensitivity and specificity