ASCC2 Antibody

What is ASCC2 and what cellular functions does it serve?

ASCC2 (Activating Signal Cointegrator 1 Complex Subunit 2) is a ubiquitin-binding protein involved in multiple critical cellular processes. It plays a significant role in DNA damage repair as a component of the ASCC complex, where it recruits ASCC3 and ALKBH3 to sites of DNA damage by binding to polyubiquitinated proteins with 'Lys-63'-linked polyubiquitin chains. ASCC2 also functions as part of the ASC-1 complex that enhances NF-kappa-B, SRF, and AP1 transactivation .

Additionally, ASCC2 is involved in the activation of the ribosome quality control (RQC) pathway, which degrades nascent peptide chains during problematic translation. It specifically recognizes and binds RPS20/uS10 ubiquitinated by ZNF598, promoting recruitment of the RQT (ribosome quality control trigger) complex on stalled ribosomes, followed by disassembly of these stalled ribosomes .

What types of ASCC2 antibodies are available for research applications?

Several types of ASCC2 antibodies are available for research purposes:

• Rabbit polyclonal antibodies against human ASCC2, such as those from Atlas Antibodies (HPA001439), Abcam (ab228827), and Proteintech (11529-1-AP) .

• These antibodies vary in their applications and validation status, with some validated for Western blot (WB), immunohistochemistry (IHC), immunofluorescence/immunocytochemistry (IF/ICC), flow cytometry (FC), and immunoprecipitation (IP) .

• The antibodies are generated using different immunogens, including recombinant fragment proteins within human ASCC2 .

When selecting an ASCC2 antibody, researchers should consider whether they need a validated in vitro diagnostic (IVD) antibody or a research use only (RUO) antibody, as the validation requirements and responsibilities differ significantly between these categories .

How do I determine which ASCC2 antibody clone is appropriate for my specific research application?

Selecting the optimal ASCC2 antibody clone requires consideration of several factors:

• Check resources like NORDIQC (https://www.nordiqc.org/) which provide information on antibody performance across different platforms and applications .

• Review the validated applications for each antibody clone. For example, Proteintech's 11529-1-AP has been validated for WB, IHC, IF/ICC, FC, and IP, while other clones may have more limited validated applications .

• Consider the specific cell or tissue types you're working with. Certain antibodies have been validated in specific samples such as HeLa cells, HepG2 cells, human colon tissue, and brain tissue .

• For challenging applications, examine published literature where specific ASCC2 antibody clones have been successfully used in experimental contexts similar to yours.

• Consider the detection method and platform compatibility. Some antibodies perform better on specific automated platforms or with particular detection systems .

The appropriate choice depends on your specific experimental goals, sample types, and detection methods. For critical experiments, testing multiple antibody clones may be necessary to identify the best performer in your specific system.

What are the recommended dilutions and detection methods for ASCC2 antibodies in different applications?

Optimal dilutions for ASCC2 antibodies vary by application and specific antibody clone. Based on available data for Proteintech's 11529-1-AP antibody, the following dilutions are recommended:

For Abcam's ab228827 antibody, slightly different dilutions have been reported: 1/5000 for WB and 1/500 for ICC/IF and IHC .

These dilutions should be considered starting points, and optimization is essential for each experimental system. Every variable including fixation method, incubation time, detection system, and sample type will affect the quality of staining and should be carefully calibrated .

What antigen retrieval methods are most effective for ASCC2 detection in immunohistochemistry?

For optimal detection of ASCC2 in immunohistochemistry applications, specific antigen retrieval methods have been recommended:

• Primary recommendation: TE buffer pH 9.0 (Tris-EDTA buffer) .

• Alternative method: Citrate buffer pH 6.0 .

The effectiveness of antigen retrieval depends on multiple factors:

• Pre-analytical variables (fixation time, processing methods)

• The specific epitope recognized by the antibody

• Tissue type being examined

• Platform and detection system used

When optimizing antigen retrieval for ASCC2 detection, researchers should consider:

• Testing both recommended buffer systems

• Adjusting retrieval times and temperatures

• Documenting results to determine optimal conditions for specific experimental contexts

The choice of antigen retrieval method can dramatically affect both sensitivity and specificity of ASCC2 detection in tissue sections . For challenging samples, it may be necessary to test a range of pH conditions and retrieval times to determine the optimal protocol.

How should researchers validate ASCC2 antibodies before implementing them in experimental workflows?

Comprehensive validation of ASCC2 antibodies is essential for ensuring reliable results. A systematic validation approach should include:

• Documentation of controls: Maintain records of positive and negative control tissues used, including validation images. This documentation is required for accreditation purposes .

• Tissue/cell line validation: Confirm reactivity with samples known to express ASCC2, such as HeLa cells, HepG2 cells, human colon tissue, and brain tissue. Include negative controls by omitting primary antibody or using tissues with minimal ASCC2 expression .

• Western blot verification:

  • Confirm detection of a band at the expected molecular weight (~86 kDa for ASCC2)

  • Verify in multiple cell lines (e.g., HeLa, HepG2, HEK-293T)

• Cross-platform validation:

  • If possible, send samples to reference laboratories for comparison

  • Compare results across different applications (e.g., WB vs. IHC vs. IF)

• Optimization of experimental conditions:

  • Test different antibody dilutions for each application

  • Optimize antigen retrieval methods for IHC

  • Determine ideal fixation protocols for ICC/IF

• Documentation of all validation steps: Create a simple but comprehensive validation document containing information about cases used, expression patterns observed, and comparison with known results or molecular data if applicable .

A well-validated antibody will produce consistent results across multiple experiments and demonstrate the expected expression pattern and molecular weight in positive control samples.

What methods can be used to study ASCC2's role in DNA repair pathways?

Investigating ASCC2's function in DNA repair requires specialized methodological approaches:

• Co-localization studies: Use validated ASCC2 antibodies in immunofluorescence to detect co-localization with known DNA damage markers after inducing DNA damage with UV radiation, ionizing radiation, or chemical agents .

• Protein interaction analysis: Employ immunoprecipitation with ASCC2 antibodies followed by Western blot to detect interactions with ASCC3, ALKBH3, and other DNA repair factors .

• Functional assays: Compare DNA repair efficiency in cells with normal ASCC2 expression versus ASCC2-depleted cells using comet assays or γH2AX foci resolution kinetics.

• Domain mapping: Use truncated ASCC2 constructs to identify which domains are essential for recruitment to DNA damage sites and interaction with polyubiquitinated proteins .

• Ubiquitin binding analysis: Investigate ASCC2's interaction with 'Lys-63'-linked polyubiquitin chains through in vitro binding assays or cellular ubiquitination studies .

These approaches can provide insights into how ASCC2 functions within the ASCC complex to facilitate DNA repair, particularly in response to specific types of DNA damage. Understanding this role may have implications for cancer research and therapeutic development.

How can ASCC2's function in ribosome quality control be experimentally investigated?

To study ASCC2's role in ribosome quality control (RQC), researchers can employ several specialized techniques:

• Ribosome profiling: Analyze ribosome occupancy on mRNAs in cells with normal versus depleted ASCC2 to identify potential translation stalling sites .

• Co-immunoprecipitation: Use ASCC2 antibodies to pull down associated proteins and detect interactions with ubiquitinated RPS20/uS10 and components of the RQT complex .

• Nascent peptide analysis: Monitor the fate of stalled translation products in ASCC2-deficient cells compared to wild-type cells using pulse-chase experiments.

• Polysome profiling: Assess the distribution of ribosomes in sucrose gradients to detect accumulation of stalled ribosomes in the absence of ASCC2.

• Ubiquitination assays: Investigate the interaction between ASCC2 and ZNF598-ubiquitinated RPS20 using in vitro reconstitution approaches .

• Fluorescence microscopy: Visualize the recruitment of ASCC2 to stalled ribosomes using fluorescently tagged ASCC2 and ribosomal markers.

Understanding ASCC2's function in ribosome quality control has implications for multiple biological processes, including proteostasis, cellular stress responses, and potentially neurodegenerative diseases associated with protein misfolding.

What are common technical challenges with ASCC2 antibodies and how can they be resolved?

Researchers working with ASCC2 antibodies may encounter several technical issues:

• Weak or inconsistent signal in Western blot:

  • Increase antibody concentration (try 1:500 instead of 1:3000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Ensure adequate protein loading (30 μg of total lysate is recommended)

  • Use fresh lysates with complete protease inhibitors

• High background in immunohistochemistry:

  • Optimize blocking conditions (longer blocking time, alternative blocking reagents)

  • Reduce antibody concentration

  • Extend washing steps

  • Test alternative antigen retrieval methods (switch between pH 9.0 TE buffer and pH 6.0 citrate buffer)

• Poor reproducibility between experiments:

  • Standardize all pre-analytical variables (fixation time, processing methods)

  • Document each step of the protocol meticulously

  • Use validated positive controls in each experiment

  • Consider automated staining platforms for consistency

• Cross-reactivity with other proteins:

  • Validate antibody specificity through multiple approaches

  • Consider using alternative antibody clones targeting different epitopes

  • Perform peptide competition assays to confirm specificity

• Inadequate signal in immunofluorescence:

  • Test different fixation methods (paraformaldehyde vs. methanol)

  • Optimize permeabilization conditions

  • Use signal amplification systems when necessary

  • Extend primary antibody incubation time

Systematic troubleshooting with careful documentation of each optimization step is essential for resolving these technical challenges and establishing reliable protocols for ASCC2 detection.

How does ASCC2 coordinate with other components of the ASCC complex in cellular response to stress?

ASCC2 plays a central coordinating role within the ASCC complex during stress responses:

• ASCC2 functions as the ubiquitin-binding component that recognizes specific ubiquitination signals at sites of DNA damage or on stalled ribosomes .

• Upon binding to ubiquitinated proteins, ASCC2 recruits other ASCC complex members including ASCC3 (an RNA helicase) and ALKBH3 (a DNA demethylase) to form a functional complex .

• This recruitment is likely regulated by stress-specific signaling pathways that may modify ASCC2 or its interaction partners through post-translational modifications.

• The coordinated activity of the ASCC complex components allows for specialized responses to different types of cellular stress, including DNA damage and translation stress .

Research questions that remain to be fully addressed include:

• How is ASCC2 recruitment to stress sites regulated?

• What determines whether ASCC2 engages in DNA repair versus ribosome quality control functions?

• How do post-translational modifications of ASCC2 affect its function in different contexts?

Understanding these coordination mechanisms could provide insights into how cells prioritize different stress responses and maintain cellular homeostasis.

What methods can be used to study potential post-translational modifications of ASCC2?

Investigating post-translational modifications (PTMs) of ASCC2 requires specialized techniques:

• Mass spectrometry-based approaches:

  • Immunoprecipitate ASCC2 using validated antibodies from cells under different conditions

  • Analyze by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Use phospho-enrichment techniques to specifically study phosphorylation

  • Compare PTM profiles between normal and stress conditions

• PTM-specific antibodies:

  • Use antibodies that recognize specific modifications (phosphorylation, ubiquitination, SUMOylation)

  • Perform Western blots of immunoprecipitated ASCC2 with these antibodies

• Site-directed mutagenesis:

  • Identify potential PTM sites through predictive algorithms or mass spectrometry

  • Generate ASCC2 mutants where these sites are altered to non-modifiable residues

  • Assess how these mutations affect ASCC2 function and interactions

• In vitro modification assays:

  • Express and purify recombinant ASCC2

  • Incubate with kinases, ubiquitin ligases, or other modifying enzymes

  • Analyze resulting modifications by Western blot or mass spectrometry

• Proximity-based labeling:

  • Use BioID or TurboID fused to ASCC2 to identify proteins in proximity

  • Determine if these include enzymes involved in post-translational modifications

Understanding ASCC2's post-translational modifications could provide critical insights into how its diverse functions are regulated in different cellular contexts and stress conditions.

How can computational approaches complement antibody-based studies of ASCC2?

Computational methods offer powerful complementary approaches to experimental ASCC2 research:

• Structural modeling and prediction:

  • Predict ASCC2 protein structure using AlphaFold or similar tools

  • Model interactions with ubiquitin chains, ASCC3, ALKBH3, and ribosomal proteins

  • Identify potential functional domains and binding interfaces

• Network analysis:

  • Construct protein-protein interaction networks centered on ASCC2

  • Identify potential new interaction partners and functional associations

  • Generate testable hypotheses about ASCC2's role in cellular pathways

• Expression correlation analysis:

  • Analyze public gene expression databases to identify genes co-regulated with ASCC2

  • Investigate expression patterns across different tissues and disease states

  • Correlate ASCC2 expression with DNA damage response or ribosome-associated genes

• Evolutionary analysis:

  • Compare ASCC2 sequences across species to identify conserved domains

  • Perform co-evolutionary analysis to predict functional interactions

  • Identify species-specific differences that might suggest specialized functions

• Mutation impact prediction:

  • Analyze how disease-associated mutations might affect ASCC2 structure and function

  • Predict the impact of experimental mutations for functional studies

  • Identify critical residues for antibody recognition

These computational approaches can guide antibody-based experimental design, help interpret experimental results, and suggest new directions for ASCC2 research that might not be immediately apparent from experimental data alone.