RNASEH2C Antibody

What is RNASEH2C and what cellular functions does it perform?

RNASEH2C is one of three subunits (along with RNASEH2A and RNASEH2B) that form the heterotrimeric RNase H2 complex. This complex functions as a ribonuclease that specifically cleaves RNA in RNA-DNA hybrids. RNASEH2C serves as a scaffolding protein within this complex, playing a crucial role in maintaining the structural integrity of RNase H2 .

The RNase H2 complex performs several vital cellular functions:

• Removal of ribonucleotides misincorporated during DNA replication

• Processing of RNA-DNA hybrids formed during DNA replication

• Involvement in DNA error repair mechanisms

• Maintenance of genomic stability

• Immune system regulation by removing unnecessary DNA fragments that might trigger immune responses

The protein has a calculated molecular weight of approximately 18 kDa (specifically 17.8 kDa), but is observed at approximately 22 kDa in Western blot analyses .

What are the established applications for RNASEH2C antibodies in research?

RNASEH2C antibodies have been validated for multiple experimental applications with the following recommended protocols:

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

How should I optimize Western blot protocols specifically for RNASEH2C detection?

Successful detection of RNASEH2C by Western blot requires specific optimization steps:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for efficient extraction

  • Include phosphatase inhibitors if studying potential phosphorylation states

  • Optimize protein loading to 20-50 μg per lane for cell lysates

• Gel selection and transfer:

  • Use 12-15% polyacrylamide gels due to RNASEH2C's relatively small size (18 kDa calculated, 22 kDa observed)

  • Use PVDF membranes with 0.2 μm pore size rather than 0.45 μm for better retention of small proteins

  • Transfer at lower voltage (30V) overnight at 4°C for efficient transfer of small proteins

• Antibody incubation:

  • Start with 1:1000 dilution for most anti-RNASEH2C antibodies, then optimize

  • For polyclonal antibodies like 16518-1-AP, begin at 1:500 dilution

  • Increase blocking time to 2 hours to reduce background

  • Use 5% BSA rather than milk for blocking and antibody dilution

• Detection controls:

  • Include lysates from RNASEH2C knockdown cells as negative controls

  • HEK-293 and HeLa cells serve as reliable positive controls

The observed molecular weight of approximately 22 kDa is slightly higher than the calculated 18 kDa, which is important to consider when identifying bands .

What controls are essential when validating RNASEH2C knockdown or knockout experiments?

For rigorous validation of RNASEH2C knockdown/knockout studies, implement these controls:

• Expression controls:

  • Verify knockdown efficiency by qRT-PCR measuring RNASEH2C mRNA levels

  • Perform Western blot using validated anti-RNASEH2C antibodies to confirm protein reduction

  • Include multiple shRNA constructs targeting different regions of RNASEH2C (as demonstrated in studies using sh2 and sh4 constructs)

• Functional controls:

  • Measure RNase H2 enzyme activity using an in vitro ribonucleotide excision repair (RER) assay

  • Use a labeled DNA oligomer containing a single ribonucleotide as substrate

  • Compare cleavage efficiency between control and knockdown samples

  • Expect approximately 70% reduction in RER activity with effective RNASEH2C knockdown

• Specificity controls:

  • Include rescue experiments with exogenous RNASEH2C expression resistant to the knockdown construct

  • Monitor levels of other RNase H2 complex subunits (RNASEH2A, RNASEH2B) to assess complex stability

  • Test for compensatory upregulation of RNASEH1, which has been observed in some RNASEH2C knockdown models

• Phenotypic validation:

  • Assess cell proliferation, as RNASEH2C deficiency may impact cell growth

  • Examine cell cycle distribution using flow cytometry

  • Evaluate DNA damage markers such as γH2AX foci formation

How can I distinguish between enzymatic and non-enzymatic functions of RNASEH2C in experimental models?

Differentiating between RNASEH2C's enzymatic and non-enzymatic roles requires a multi-faceted experimental approach:

• Comparative knockdown strategy:

  • Perform parallel knockdown of RNASEH2C and RNASEH2A (catalytic subunit)

  • Compare phenotypic outcomes between these knockdowns

  • Differential effects suggest non-enzymatic functions of RNASEH2C

• Structure-function rescue experiments:

  • Generate RNASEH2C mutants that maintain complex formation but impair enzymatic activity

  • Express these mutants in RNASEH2C-knockdown cells

  • Assess which phenotypes are rescued despite reduced enzymatic activity

  • Research has shown that RNASEH2C's role in metastasis is independent of RNase H2 enzymatic activity

• Biochemical activity assessment:

  • Purify RNase H2 complexes containing wild-type or mutant RNASEH2C

  • Measure enzyme activity using in vitro assays with RNA:DNA hybrid substrates

  • Quantify differences in catalytic efficiency

  • AGS-causing mutations in RNASEH2C can reduce activity while maintaining complex formation

• Protein-protein interaction studies:

  • Perform immunoprecipitation followed by mass spectrometry to identify RNASEH2C-specific interactors

  • Compare interactomes between wild-type and enzymatically-impaired complexes

  • Focus on interactions independent of RNase H2 activity

Research has established that RNASEH2C's role in breast cancer metastasis functions through a non-enzymatic mechanism involving T cell-mediated immune responses, distinct from its enzymatic role in the RNase H2 complex .

What methodologies should I employ to investigate RNASEH2C's role in cancer metastasis?

Based on recent research identifying RNASEH2C as a metastasis susceptibility factor, these methodologies are recommended:

• In vivo metastasis models:

  • Establish orthotopic mammary fat pad injections using cell lines with modulated RNASEH2C expression

  • Quantify both primary tumor growth and spontaneous pulmonary metastases

  • Normalize metastasis counts to primary tumor size

  • Perform experimental (tail vein) metastasis assays to focus specifically on late-stage metastatic steps

  • Use multiple cancer cell lines (e.g., Mvt1 and 4T1) to ensure results aren't cell line-specific

• Immune component analysis:

  • Compare metastatic outcomes in immunocompetent versus immunodeficient (e.g., athymic nude) mice

  • Perform immunophenotyping of tumor-infiltrating immune cells

  • Use flow cytometry to quantify specific immune cell populations:

    • CD8+ IFN-γ+ (activated cytotoxic T cells)

    • CD4+ Foxp3+ (regulatory T cells)

    • NK cells

  • Target tissues should include primary tumors, metastatic sites, and spleens

• Molecular pathway investigation:

  • Conduct RNA-sequencing of tumors with different RNASEH2C expression levels

  • Apply pathway analysis to identify affected signaling networks

  • Examine immune-related pathways specifically

  • Investigate cGAS-STING pathway activation, which is implicated in RNase H2 deficiency but may show context-dependent activation

• Clinical correlation:

  • Analyze RNASEH2C expression in patient samples with different metastatic outcomes

  • Correlate expression with immune cell infiltration patterns

  • Consider RNASEH2C as part of a gene panel for metastasis risk prediction

Research has shown that knocking down RNASEH2C significantly reduced metastatic burden in multiple breast cancer models, and this effect was dependent on T cell-mediated immunity .

How should I approach studying the relationship between RNASEH2C dysfunction and Aicardi-Goutières syndrome?

Investigating RNASEH2C's role in Aicardi-Goutières syndrome (AGS) requires specialized approaches:

• Patient-derived cell model development:

  • Establish lymphoblastoid cell lines from AGS patients with defined RNASEH2C mutations

  • Create isogenic cell lines using CRISPR/Cas9 to introduce specific mutations

  • Compare with cells carrying mutations in other RNase H2 subunits to identify RNASEH2C-specific effects

• RNase H2 complex analysis:

  • Assess complex formation using co-immunoprecipitation assays

  • Determine stability of mutant complexes under varying conditions

  • Measure enzyme activity using ribonucleotide excision repair (RER) assays

  • Research shows most AGS-causing RNASEH2C mutations permit complex formation but may reduce stability or activity

• Nucleic acid metabolism assessment:

  • Quantify accumulation of ribonucleotides in genomic DNA

  • Detect RNA:DNA hybrids using S9.6 antibody staining

  • Measure DNA damage markers like γH2AX foci

  • Assess post-replication repair activation by monitoring PCNA ubiquitylation

• Immune pathway investigation:

  • Monitor activation of the cGAS-STING pathway

  • Measure type I interferon production

  • Assess expression of interferon-stimulated genes

  • Compare immune signatures between AGS patient cells and cancer cells with RNASEH2C modulation

Unlike in breast cancer metastasis, where RNASEH2C's role appears independent of enzymatic activity, AGS pathology is typically linked to reduced RNase H2 enzymatic function leading to accumulation of nucleic acids that trigger immune responses .

What are the critical validation steps to ensure RNASEH2C antibody specificity?

Ensuring antibody specificity is crucial for reliable results in RNASEH2C research:

• Genetic validation approaches:

  • Test antibody on samples with RNASEH2C knockdown or knockout

  • Verify absence or significant reduction of signal in Western blot, IHC, or IF

  • Include positive controls from tissues known to express RNASEH2C (e.g., spleen, testis)

• Cross-reactivity assessment:

  • Test antibody against recombinant RNASEH2C protein

  • Verify lack of cross-reactivity with other RNase H2 subunits

  • Perform peptide competition assays to confirm binding specificity

  • Check reactivity across species if performing comparative studies

• Application-specific validation:

  • For Western blot: Verify single band at expected molecular weight (22 kDa)

  • For IHC/IF: Compare staining pattern with established RNASEH2C localization (primarily nuclear)

  • For IP: Confirm enrichment of RNASEH2C and known interaction partners

  • Validate across multiple cell/tissue types to account for expression variations

• Multiple antibody concordance:

  • Compare results using antibodies targeting different epitopes of RNASEH2C

  • Polyclonal antibodies like 16518-1-AP detect the entire protein, while others may target specific domains

  • Consistent results across antibodies increase confidence in specificity

For immunohistochemistry applications specifically, suggested antigen retrieval with TE buffer pH 9.0 improves specificity, though citrate buffer pH 6.0 may be used as an alternative .

How do I interpret contradictory findings regarding RNASEH2C function in different experimental systems?

Resolving contradictions in RNASEH2C research requires careful consideration of experimental context:

When facing contradictory results, carefully evaluate differences in experimental systems, knockdown/mutation strategies, and specific functions being assessed.

How might advanced protein design approaches be applied to develop novel anti-RNASEH2C research tools?

Recent advances in protein design, particularly in antibody engineering, offer promising approaches for developing next-generation RNASEH2C research tools:

• De novo antibody design:

  • New computational methods like fine-tuned RFdiffusion networks can design antibodies targeting specific epitopes

  • Apply these methods to develop antibodies against distinctive domains of RNASEH2C

  • Target epitopes that distinguish between RNASEH2C's enzymatic and non-enzymatic functions

  • Design antibodies that specifically recognize disease-associated RNASEH2C conformations

• Structure-based epitope targeting:

  • Utilize known structural information about the RNase H2 complex

  • Design antibodies targeting interfaces between RNASEH2C and other complex components

  • Create tools that selectively disrupt specific protein-protein interactions

  • This approach could help isolate RNASEH2C functions independent of the RNase H2 complex

• Intrabody development:

  • Design antibody fragments that function inside cells (intrabodies)

  • Target specific subcellular pools of RNASEH2C

  • Use these tools to modulate RNASEH2C function in specific cellular compartments

  • This approach could help dissect RNASEH2C's role in different cellular processes

• Conditional detection systems:

  • Develop antibody-based biosensors that detect RNASEH2C only when engaged in specific functions

  • Create proximity-based reporters that illuminate when RNASEH2C interacts with specific partners

  • These tools could help visualize dynamic aspects of RNASEH2C function in living cells

These approaches could overcome current limitations in studying RNASEH2C function by providing more precise tools for manipulating and monitoring specific aspects of its activity.

What experimental approaches would best elucidate the mechanisms linking RNASEH2C to T cell-mediated immune responses in cancer?

Based on recent discoveries of RNASEH2C's role in modulating T cell responses in cancer, these experimental approaches would advance our understanding:

• Single-cell analysis of tumor immune microenvironment:

  • Perform single-cell RNA sequencing of tumors with different RNASEH2C expression levels

  • Map changes in immune cell populations and their activation states

  • Identify cell-type specific transcriptional responses to RNASEH2C modulation

  • Trace intercellular communication pathways between cancer cells and immune cells

• Cancer cell-T cell co-culture systems:

  • Establish in vitro co-culture systems with cancer cells and T cells

  • Modulate RNASEH2C expression in cancer cells and measure T cell activation markers

  • Identify tumor-derived factors mediating T cell activation

  • Use transwell systems to distinguish contact-dependent from soluble factor-mediated effects

• Antigen presentation and recognition studies:

  • Investigate whether RNASEH2C affects tumor antigen processing/presentation

  • Analyze MHC class I peptide repertoire in cells with different RNASEH2C levels

  • Assess T cell receptor (TCR) diversity in responding T cells

  • Determine if RNASEH2C knockdown exposes neo-antigens or enhances presentation of existing antigens

• Signaling pathway dissection:

  • Examine cytokine/chemokine profiles in the tumor microenvironment

  • Assess activation of pathways that bridge innate and adaptive immunity

  • Determine if RNASEH2C knockdown activates alternative nucleic acid sensing pathways beyond cGAS-STING

  • Identify downstream transcription factors mediating immune gene expression changes