C1orf112 Antibody

What is C1orf112 and why is it important to study?

C1orf112 (Chromosome 1 open reading frame 112) is a protein that has recently garnered attention for its role in DNA repair pathways. It functions as a novel regulator of interstrand crosslink (ICL) repair and is associated with homology-directed DNA repair pathways . Notably, C1orf112 forms a complex with the N-terminal domain of the AAA+ ATPase FIGNL1 through its WCF motif and regulates FIGNL1's interaction with RAD51, thereby influencing DNA repair mechanisms . Research indicates that C1orf112 is essential for mammalian development, as homozygous knockout mice are not viable . The protein appears to be highly conserved throughout eukaryotes, from plants to humans, with particularly high conservation in primates .

What applications are C1orf112 antibodies validated for?

Current commercially available C1orf112 antibodies have been validated for several research applications:

• Immunohistochemistry on paraffin-embedded sections (IHC-P)

• Western blotting (WB)

• Immunocytochemistry/Immunofluorescence (ICC-IF)

These validations provide researchers with reliable tools to investigate C1orf112 expression, localization, and function across different experimental setups.

What tissues should be used as positive controls for C1orf112 antibody validation?

Based on expression data, the following tissues serve as excellent positive controls:

• Testis (highest expression)

• Lymph nodes

• Human liver cancer tissue

• Cerebellum

Gene expression analyses from multiple individuals across 27 different tissues have consistently shown that C1orf112 is expressed most highly in testes, followed by lymph nodes, bone marrow, and cerebellum . For cancer-related studies, human liver cancer tissue has been specifically recommended as a positive control for IHC applications .

How can C1orf112 antibodies be used to investigate DNA damage repair pathways?

C1orf112 antibodies provide powerful tools for studying DNA damage repair mechanisms through several approaches:

• Colocalization studies: By combining C1orf112 antibodies with markers of DNA damage (γ-H2AX) or DNA repair proteins (RAD51, FANCD2), researchers can investigate whether C1orf112 is recruited to DNA damage sites .

• Chromatin immunoprecipitation (ChIP): C1orf112 antibodies can be used to determine if the protein associates with damaged DNA regions.

• Immunoprecipitation coupled with mass spectrometry: This approach has revealed 31 physical protein-protein interactions for C1orf112, providing insights into its functional networks .

• Immunofluorescence after DNA damage induction: C1orf112 antibodies can be used to track protein dynamics following treatment with DNA damaging agents such as cisplatin, which is particularly relevant since C1orf112 depletion increases sensitivity to interstrand crosslinking agents .

Research has shown that C1orf112 depletion increases both the number and intensity of γ-H2AX foci in cells, particularly after cisplatin treatment, suggesting a role in preventing replication stress-induced DNA breaks .

What is the relationship between C1orf112 expression and cancer that can be studied using these antibodies?

C1orf112 antibodies are valuable tools for investigating the protein's role in cancer through several research approaches:

• Expression analysis in tumor tissues: C1orf112 has been found to be overexpressed in various cancers compared to healthy tissues . Antibodies can help quantify this differential expression across tumor types.

• Correlation with TP53 status: Studies have shown a twofold down-regulation of C1orf112 in cells with mutant TP53 compared to wild-type TP53 in invasive bladder tumor cells . Antibodies can help validate these findings at the protein level.

• Prognostic marker evaluation: C1orf112 has been identified as part of gene expression signatures associated with disease outcomes in gastric cancer patients . Immunohistochemistry using validated antibodies could help establish its value as a prognostic marker.

• Cell cycle regulation studies: C1orf112 is co-expressed with several cell cycle regulators, and antibodies can be used to study its dynamics throughout the cell cycle and potential contributions to dysregulated proliferation in cancer cells .

How does C1orf112 contribute to genomic stability, and how can antibodies help elucidate this function?

C1orf112 appears to play a crucial role in maintaining genomic stability through multiple mechanisms that can be investigated using specific antibodies:

• DNA damage quantification: Research has shown that C1orf112 depletion significantly increases both the number and intensity of γ-H2AX foci in cells, even without treatment with DNA damaging agents, indicating a role in protecting against endogenous DNA damage .

• Protein complex analysis: C1orf112, through its WCF motif, forms a complex with FIGNL1 and regulates its interaction with RAD51 . Antibodies can be used in co-immunoprecipitation experiments to study these interactions.

• Mitotic spindle assembly: More recent findings suggest that C1orf112 regulates PLK1 kinase activity at kinetochores and promotes faithful chromosome segregation in prometaphase . Immunofluorescence with C1orf112 antibodies can help visualize its localization during mitosis.

• Homologous recombination studies: C1orf112 (also called FIRRM) in complex with FIGL1 may regulate homologous recombination . Antibodies can help track its involvement in this critical DNA repair pathway.

What are the recommended protocols for immunohistochemistry with C1orf112 antibodies?

For optimal results when using C1orf112 antibodies in immunohistochemistry:

• Sample preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissue sections.

• Antigen retrieval: Heat-mediated antigen retrieval with citrate buffer (pH 6.0) is recommended .

• Recommended dilutions:

  • For IHC: 1:100-1:300 dilution

  • For IHC-P: 1:500 dilution has been validated

• Primary antibody incubation: Typically overnight at 4°C or 1-2 hours at room temperature.

• Detection system: An appropriate HRP-conjugated secondary antibody system matching the host species (typically rabbit for available C1orf112 antibodies) .

• Positive controls: Human liver cancer tissue, testis, or other tissues with known high C1orf112 expression should be included .

What factors should be considered when selecting a C1orf112 antibody for research?

When selecting a C1orf112 antibody for specific research applications, consider:

• Immunogen information: Current antibodies are raised against different regions of the protein:

  • Some target the region within amino acids 650-800

  • Others use fusion proteins of human C1orf112

• Validation data: Review available validation data for your specific application. Currently available antibodies are primarily validated for IHC-P, with some also validated for ICC-IF and WB .

• Clonality: Currently, most available C1orf112 antibodies are rabbit polyclonal antibodies , which may provide broader epitope recognition but potentially more batch-to-batch variation.

• Storage and stability: These antibodies are typically stored at -20°C with glycerol as a cryoprotectant and have a stability of approximately 1 year .

• Background considerations: For tissues with potential non-specific staining, additional blocking steps may be necessary.

How can researchers validate the specificity of C1orf112 antibodies?

To ensure antibody specificity for reliable research results:

• Positive and negative controls: Include tissues with known C1orf112 expression patterns (high in testis, low in other tissues) and test the antibody in C1orf112 knockout or knockdown models.

• Western blot validation: Confirm the antibody detects a protein of the expected molecular weight (~96.6 kDa for the precursor) .

• Competing peptide assay: Pre-incubate the antibody with the immunizing peptide to demonstrate that staining is blocked when the antibody binding sites are occupied.

• siRNA knockdown: Validate that antibody signal decreases proportionally following siRNA-mediated reduction of C1orf112.

• Multiple antibody comparison: When possible, compare staining patterns from antibodies targeting different epitopes of C1orf112.

What are common challenges when working with C1orf112 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with C1orf112 antibodies:

• Low signal intensity: This may occur due to low expression levels in certain tissues or cell types.

  • Solution: Optimize antibody concentration, increase incubation time, enhance antigen retrieval, or use signal amplification systems.

• Background staining: Non-specific binding can complicate interpretation of results.

  • Solution: Increase blocking time, use additional blocking agents, or optimize antibody dilution.

• Variability between experiments: Polyclonal antibodies may show batch-to-batch variation.

  • Solution: Purchase sufficient quantity for complete studies, include consistent positive controls, and standardize protocols.

• Detection of multiple isoforms: C1orf112 has 17 mRNA variants encoding 15 functional proteins .

  • Solution: Be aware of which isoforms your antibody detects and interpret results with this understanding.

How can researchers effectively use C1orf112 antibodies to study protein-protein interactions?

To investigate C1orf112 protein interactions using antibodies:

• Co-immunoprecipitation: C1orf112 antibodies can be used to pull down the protein along with its interaction partners, which can then be identified by mass spectrometry or western blotting. This approach has revealed interactions with proteins like FIGNL1 and PPP1CC .

• Proximity ligation assay (PLA): This technique can visualize and quantify protein-protein interactions in situ, allowing researchers to study C1orf112 interactions with proteins like RAD51 or FIGNL1 in their native cellular context.

• Immunofluorescence co-localization: Dual labeling with C1orf112 antibodies and antibodies against potential interaction partners can provide evidence of spatial proximity.

• FRET/BRET analysis: When combined with fluorescently tagged potential partners, C1orf112 antibodies can be used in acceptor photobleaching FRET to study protein interactions.

What emerging applications for C1orf112 antibodies show promise for advancing cancer research?

Several emerging applications of C1orf112 antibodies could significantly impact cancer research:

• Biomarker development: C1orf112 has been identified as part of gene signatures associated with gastric cancer outcomes . Antibody-based assays could help develop it as a clinical biomarker.

• Therapeutic target validation: As C1orf112 depletion sensitizes cells to cisplatin and PARP inhibitors , antibodies can help validate it as a potential therapeutic target in combination treatments.

• Prognostic stratification: Immunohistochemistry panels including C1orf112 might help stratify patients for appropriate treatment regimens, particularly in cancers where DNA repair deficiencies guide therapy.

• Cell cycle checkpoint analysis: Given C1orf112's association with mitosis and chromosome segregation , antibodies could help elucidate its role in cell cycle checkpoints that are frequently dysregulated in cancer.

How might C1orf112 antibodies contribute to understanding rare genetic disorders?

C1orf112 antibodies could be valuable tools for investigating several genetic disorders:

• Fanconi anemia: Given C1orf112's role in interstrand crosslink repair and its functional relationship with the Fanconi anemia pathway , antibodies could help elucidate disease mechanisms and identify potential therapeutic targets.

• Developmental disorders: Since C1orf112 knockout mice are not viable , antibodies could help investigate its role in human developmental disorders by studying expression patterns in appropriate tissues.

• Cancer predisposition syndromes: In syndromes characterized by deficiencies in DNA repair (like hereditary breast and ovarian cancer syndrome), C1orf112 antibodies could help investigate compensatory mechanisms or synthetic lethal interactions.

• Mitotic defect disorders: Given its role in chromosome segregation , C1orf112 antibodies might help understand disorders characterized by chromosomal instability.