C1orf123 Antibody

What is C1orf123 protein and what are its basic characteristics?

C1orf123, also known as CZIB, is a human protein encoded by an open reading frame located on chromosome 1. The full-length protein consists of 160 amino acids resulting from the splicing of 8 exons. It belongs to the DUF866 (Domain of Unknown Function 866) superfamily, which is exclusively found in eukaryotic cells. The protein has an observed molecular weight of approximately 26 kDa and is characterized by a UniProt ID of Q9NWV4 and NCBI Gene ID of 54987 .

C1orf123 has three known isoforms: the canonical 160-amino acid form (NP_060357.1), isoform 2 consisting of 143 amino acids (NP_001291688.1), and isoform 3 with 113 amino acids (NP_001291689.1). The shorter isoforms lack one and two alternate in-frame exons at the 5' end, respectively .

The crystal structure of C1orf123 reveals a distinctive 2-fold internal symmetry that divides the monomeric protein into two mirrored halves with distinct electrostatic potential. The N-terminal half includes a zinc-binding domain that interacts with a zinc ion near a potential ligand binding cavity .

What applications are C1orf123 antibodies validated for?

C1orf123 antibodies from Proteintech (such as 84665-4-PBS and 84665-4-RR) have been validated for several research applications:

• Western Blot (WB): The antibodies perform well at dilution ranges of 1:2000-1:10000

• Indirect ELISA: Both antibody formulations are suitable for this application

These antibodies have been specifically tested for reactivity with human samples and show positive detection in multiple human cell lines including Jurkat cells, K-562 cells, MOLT-4 cells, and U-937 cells .

What are the optimal storage conditions for C1orf123 antibodies?

The optimal storage conditions vary depending on the specific formulation of the C1orf123 antibody:

For the 84665-4-RR formulation, aliquoting is unnecessary for -20°C storage. The 20μl size contains 0.1% BSA as a stabilizing agent .

What is the recommended protocol for Western blot analysis using C1orf123 antibodies?

For optimal Western blot results with C1orf123 antibodies, researchers should follow these methodological steps:

• Sample preparation: Prepare lysates from appropriate human cell lines (validated cell lines include Jurkat, K-562, MOLT-4, and U-937 cells)

• Protein separation: Use standard SDS-PAGE to separate proteins

• Transfer: Transfer proteins to an appropriate membrane

• Blocking: Block nonspecific binding sites using standard blocking buffer

• Primary antibody incubation: Dilute C1orf123 antibody within the recommended range (1:2000-1:10000)

• Detection: Use appropriate secondary antibodies and detection methods

• Analysis: The target protein should be detected at approximately 26 kDa

It's advisable to optimize the antibody dilution for specific experimental conditions, as the optimal concentration may be sample-dependent. Researchers should consult the detailed protocol available from the manufacturer for specific buffer compositions and incubation times .

How should researchers design immunoprecipitation experiments with C1orf123 antibodies?

Based on published research methodologies, immunoprecipitation (IP) experiments for C1orf123 can be designed as follows:

• Antibody validation: First verify that the anti-C1orf123 antibody reacts with recombinant C1orf123 (rC1ORF123) by Western blot

• Sample preparation: Prepare cell lysates in appropriate binding buffer (e.g., 0.025M Tris, 0.15M NaCl, 0.001M EDTA, 1% NP40, 5% glycerol)

• Complex formation: Incubate the cell lysate with anti-C1orf123 antibody (approximately 6 μg per sample) overnight at 4°C to form antigen/antibody complexes

• Bead preparation: Pre-equilibrate protein A/G magnetic beads and add to the mixture

• Incubation: Incubate at room temperature with gentle rolling for 1 hour

• Washing: Remove non-specifically bound proteins through washing steps

• Elution: Elute C1orf123 and its interacting partners using an elution buffer (e.g., glycine pH 2)

• Neutralization: Neutralize the eluate with Tris pH 8.5

• Analysis: Analyze the precipitated proteins using appropriate methods such as Western blot or mass spectrometry

For enhanced protein partner identification, researchers can include a sample with added purified recombinant C1orf123 protein to enrich for interacting partners .

How can researchers investigate the potential role of C1orf123 in post-translational modifications?

C1orf123 has been identified as an interactor with O-GlcNAc Transferase (OGT), suggesting a role in post-translational modifications . To investigate this function, researchers might:

• Co-immunoprecipitation assays: Perform co-IP experiments using anti-C1orf123 antibodies to pull down OGT and other potential interacting partners involved in post-translational modifications

• O-GlcNAcylation analysis: Examine whether C1orf123 itself is subject to O-GlcNAcylation or if it modulates the O-GlcNAcylation of other proteins

• Site-directed mutagenesis: Create mutants of C1orf123, particularly in regions important for protein-protein interactions, to determine which domains are critical for its interaction with OGT

• Functional assays: Measure changes in O-GlcNAcylation patterns following C1orf123 knockdown or overexpression

• Mass spectrometry analysis: Use LC-MS/MS to identify specific sites of modification and protein interactions, similar to the methods described in the published research on C1orf123

These approaches should be complemented with appropriate controls, including IgG control experiments to identify false-positive interactions in immunoprecipitation studies .

What methods are appropriate for studying the zinc-binding properties of C1orf123?

The crystal structure of C1orf123 reveals a zinc-binding domain in the N-terminal half of the protein . To investigate the functional significance of this domain, researchers might employ:

• Site-directed mutagenesis: Introduce mutations in the CXXC motifs (CX₂CX₃₀CX₂C) that form the zinc-binding domain to assess its functional importance

• Metal chelation experiments: Use chelating agents to remove zinc and observe functional consequences

• Structural analysis: Employ X-ray crystallography or NMR to analyze structural changes upon zinc binding/removal

• Zinc-binding assays: Use spectroscopic methods to quantify zinc binding and determine binding constants

• Conformational studies: Investigate conformational changes triggered by zinc binding, particularly around the nearby ligand binding cavity identified in the crystal structure

• Comparative analysis: Compare the zinc-binding domain of C1orf123 with similar domains in other proteins, such as the C-terminal domain of human RIG-I-like receptor LGP2 mentioned in the research

These approaches can help establish the importance of zinc binding for the structural integrity and functional activity of C1orf123.

How should researchers design experiments to investigate C1orf123's potential role in neurological disorders?

Research indicates potential involvement of C1orf123 in neurological processes, with altered expression in schizophrenia, bipolar disorder, and other brain-related conditions . To investigate these connections, researchers might:

• Expression analysis: Compare C1orf123 expression levels in neurological tissue samples from patients with schizophrenia or bipolar disorder versus controls, focusing on the hippocampus where expression differences have been observed

• Animal models: Study C1orf123 expression in relevant animal models, such as methamphetamine-treated rats or models of sleep disorders where C1orf123 homologues have shown differential expression

• Cell culture studies: Utilize neuronal cell lines or primary neurons to assess the effects of C1orf123 knockdown or overexpression on neuronal function

• Synaptic localization studies: Investigate whether C1orf123 localizes to synapses, as suggested by its presence in the electric organ of Torpedo californica along with proteins related to neuromuscular junctions and presynapsis

• Genetic association studies: Examine possible genetic variations in C1orf123 that might correlate with neurological disorders

• Immunohistochemistry: Use C1orf123 antibodies for brain tissue staining to determine regional and cellular localization patterns

These approaches should include appropriate controls and validation steps to ensure the specificity of C1orf123 detection.

What are common issues with Western blot detection of C1orf123 and how can they be resolved?

When working with C1orf123 antibodies in Western blot applications, researchers might encounter several challenges:

• Multiple bands: C1orf123 has three known isoforms (160, 143, and 113 amino acids) , which may appear as multiple bands. Resolution:

  • Use positive controls with known expression of specific isoforms

  • Consider isoform-specific antibodies if available

  • Compare observed band patterns with expected molecular weights (the main isoform appears at approximately 26 kDa)

• Weak signal: If detection signal is weak despite known expression. Resolution:

  • Optimize antibody concentration within the recommended range (1:2000-1:10000)

  • Increase protein loading

  • Extend primary antibody incubation time or temperature

  • Ensure sample preparation preserves the protein of interest

• Non-specific binding: If multiple non-specific bands appear. Resolution:

  • Increase blocking time or concentration

  • Optimize washing steps

  • Dilute primary antibody further

  • Consider different blocking reagents

How can researchers distinguish between the different isoforms of C1orf123?

To differentiate between the three known isoforms of C1orf123:

• Gel resolution: Use higher percentage polyacrylamide gels (12-15%) to better separate the isoforms based on their different molecular weights

• Isoform-specific antibodies: If available, use antibodies that specifically recognize unique regions of each isoform

• RT-PCR: Design primers that can distinguish between the different transcripts

• Mass spectrometry: Use proteomic approaches to identify isoform-specific peptides

• Recombinant standards: Run purified recombinant versions of each isoform as size standards

• Tissue-specific controls: Use tissues or cell lines with known expression patterns of specific isoforms

The isoforms differ primarily at the 5' end, with isoforms 2 and 3 lacking one and two alternate in-frame exons, respectively .

What are promising avenues for future research on C1orf123 function?

Based on current knowledge, several promising research directions for C1orf123 include:

• Mitochondrial function: Investigate the suggested role of C1orf123 in mitochondrial oxidative phosphorylation

• Neurological disorders: Further explore the connections to schizophrenia, bipolar disorder, and methamphetamine effects, where altered C1orf123 expression has been observed

• Structural biology: Characterize the ligand binding cavity identified near the zinc-binding domain to identify potential natural ligands

• Post-translational modifications: Expand on the interaction with O-GlcNAc Transferase to understand the functional consequences of this interaction

• Synaptic function: Investigate the potential role in synapse structure and maintenance, as suggested by studies in Torpedo californica

• Reproductive biology: Explore the significance of high C1orf123 transcript numbers in oocytes of Polycystic Ovarian Syndrome patients

• Evolutionary studies: Examine the 2-fold internal symmetry of the protein structure, which suggests functional evolution via gene duplication

These research directions may help elucidate the physiological role of this conserved eukaryotic protein.

What emerging technologies might advance our understanding of C1orf123?

Several cutting-edge technologies could significantly advance C1orf123 research:

• CRISPR/Cas9 gene editing: Generate knockout or knock-in models to study C1orf123 function in vivo

• Single-cell transcriptomics: Examine cell-specific expression patterns in different tissues and disease states

• Cryo-EM: Obtain high-resolution structures of C1orf123 in complex with interacting partners

• Proximity labeling (BioID, APEX): Identify proteins in close proximity to C1orf123 in living cells

• Integrative multi-omics: Combine proteomics, transcriptomics, and metabolomics to understand C1orf123 function in a systems biology context

• Patient-derived iPSCs: Study C1orf123 in neuronal cells derived from patients with disorders showing altered C1orf123 expression

• High-content screening: Identify small molecules that modulate C1orf123 function or interaction with partners

Combining these technologies with the specific C1orf123 antibodies discussed here will provide researchers with powerful tools to elucidate the function of this protein.