Recombinant Human Putative uncharacterized protein C1orf98 (C1orf98)

What is Recombinant Human Putative uncharacterized protein C1orf98 and why is it significant for research?

Recombinant Human Putative uncharacterized protein C1orf98 (C1orf98) is a protein with currently unknown function that has been identified in the human genome. It is also known by the target name LINC00862 in some databases . The significance of studying uncharacterized proteins like C1orf98 lies in their potential to reveal novel cellular mechanisms, pathways, or functions that could be important for understanding human biology and disease.

Uncharacterized proteins represent significant gaps in our knowledge of the human proteome. By studying these proteins systematically, researchers contribute to completing our understanding of cellular processes. Similar to the approach used with C17orf80 (another uncharacterized protein), research on C1orf98 may eventually reveal unexpected associations with critical cellular structures or functions .

What are the basic structural and biochemical properties of C1orf98?

Based on available data, C1orf98 is identified in the UniProt database with the accession number A6NCI5 . The commercially available recombinant form is produced in mammalian cells and has a purity of >85% as determined by SDS-PAGE analysis . The protein is available in partial length rather than full-length form .

For researchers interested in C1orf98's biochemical properties, standard analytical techniques should be employed:

• Size determination through gel filtration chromatography

• Secondary structure analysis through circular dichroism

• Stability assessment through thermal shift assays

• Post-translational modification analysis through mass spectrometry

These basic characterizations provide the foundation for more advanced functional studies.

How should I design initial experiments to begin characterizing C1orf98?

Initial characterization experiments should follow a systematic approach:

• Expression analysis: Determine tissue distribution and expression levels of endogenous C1orf98 using RT-qPCR and western blotting

• Subcellular localization: Perform immunofluorescence microscopy and subcellular fractionation, similar to techniques used for C17orf80 localization

• Sequence analysis: Conduct bioinformatic analysis to identify conserved domains, motifs, or sequence similarities with characterized proteins

• Interaction studies: Perform pull-down assays and co-immunoprecipitation to identify binding partners

Table 1: Recommended Initial Characterization Experiments for C1orf98

How can I design rigorous experiments to determine C1orf98 function using true experimental research design principles?

When designing experiments to determine C1orf98 function, employ true experimental research design principles with proper controls and randomization:

• Clearly define variables: Identify independent variables (e.g., presence/absence of C1orf98, expression levels) and dependent variables (e.g., cellular phenotypes, molecular readouts)

• Establish control groups: Include appropriate negative controls (e.g., non-targeting siRNA) and positive controls (e.g., siRNA targeting a gene with known phenotype)

• Randomize samples: Ensure random distribution of experimental units to minimize bias

• Control extraneous variables: Standardize experimental conditions to isolate the effect of C1orf98 manipulation

For knockdown/knockout experiments:

• Use multiple siRNAs/sgRNAs targeting different regions of C1orf98

• Validate knockdown/knockout efficiency at protein and mRNA levels

• Perform rescue experiments by re-expressing siRNA-resistant C1orf98 constructs

These approaches will help establish causality between C1orf98 and observed phenotypes, adhering to true experimental design principles .

What methodologies are most effective for investigating protein-protein interactions involving uncharacterized proteins like C1orf98?

For investigating protein-protein interactions involving C1orf98:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express tagged C1orf98 (e.g., FLAG, HA, or BioID) in relevant cell lines

  • Perform pull-down experiments followed by mass spectrometry

  • Compare with control samples to identify specific interactors

• Proximity labeling approaches:

  • Generate BioID or APEX2 fusion constructs with C1orf98

  • Identify proteins in close proximity through biotinylation

  • Similar to approaches used for C17orf80 characterization

• Co-immunoprecipitation with candidate interactors:

  • Based on bioinformatics predictions or preliminary screens

  • Validate using both overexpressed and endogenous proteins

• Yeast two-hybrid screening:

  • Use C1orf98 as bait to screen against human cDNA libraries

  • Validate hits with orthogonal methods

These interaction studies should be performed under different cellular conditions (e.g., stress, different growth phases) to identify context-dependent interactions.

How can I resolve conflicting experimental results when studying uncharacterized proteins like C1orf98?

When facing conflicting experimental results with C1orf98:

• Systematically evaluate methodology differences:

  • Examine protein preparation methods (recombinant vs. endogenous)

  • Compare cell types or tissues used across studies

  • Assess differences in experimental conditions or reagents

• Conduct dose-response studies:

  • Test multiple concentrations or expression levels of C1orf98

  • Evaluate if effects are concentration-dependent or exhibit thresholds

• Temporal analysis:

  • Investigate if contradictory results might be due to time-dependent effects

  • Perform time-course experiments to capture dynamic processes

• Multi-method validation:

  • Apply orthogonal techniques to validate findings

  • For example, complement microscopy with biochemical fractionation

• Statistical reassessment:

  • Increase sample sizes to enhance statistical power

  • Implement more rigorous statistical analyses, including testing for outliers

Remember that contradictions can reveal important biological nuances and should be thoroughly investigated rather than dismissed.

What are the optimal storage conditions for maintaining C1orf98 stability and activity?

The optimal storage conditions for C1orf98 depend on its formulation :

• Lyophilized form:

  • Store at -20°C or -80°C

  • Expected shelf life: approximately 12 months

  • Protect from moisture and avoid repeated freeze-thaw cycles

• Liquid form:

  • Store at -20°C or -80°C

  • Expected shelf life: approximately 6 months

  • Aliquot to minimize freeze-thaw cycles

• Working aliquots:

  • Can be stored at 4°C for up to one week

  • Repeated freezing and thawing is not recommended

The stability of the protein is influenced by buffer composition, storage temperature, and the intrinsic properties of the protein itself . For long-term experiments, it's advisable to establish activity benchmarks at the beginning of the study to monitor potential degradation over time.

What is the recommended protocol for reconstituting C1orf98 for experimental use?

For reconstituting C1orf98:

• Initial preparation:

  • Briefly centrifuge the vial prior to opening to bring the contents to the bottom

  • This minimizes protein loss and ensures accurate reconstitution

• Reconstitution procedure:

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Allow the protein to dissolve completely by gentle mixing

  • Avoid vigorous vortexing that could denature the protein

• Stabilization with glycerol:

  • Add glycerol to a final concentration of 5-50%

  • 50% glycerol is the manufacturer's default recommendation

  • Glycerol serves as a cryoprotectant to maintain protein stability during freeze-thaw cycles

• Aliquoting:

  • Prepare small single-use aliquots to avoid repeated freeze-thaw cycles

  • Label clearly with concentration, date, and buffer composition

This protocol maximizes protein stability and minimizes degradation during experimental workflows.

What approaches can be used to determine the subcellular localization of C1orf98?

Determining the subcellular localization of C1orf98 requires a multi-method approach:

• Fluorescence microscopy techniques:

  • Immunofluorescence using validated antibodies against endogenous C1orf98

  • Expression of fluorescently tagged C1orf98 (with careful validation that tagging doesn't affect localization)

  • Co-localization with known organelle markers

• Biochemical fractionation:

  • Differential centrifugation to separate subcellular compartments

  • Western blotting of fractions to detect C1orf98

  • Comparison with known compartment markers (e.g., GAPDH for cytosol, Lamin A/C for nucleus)

• Proximity labeling approaches:

  • Similar to those used for characterizing C17orf80's association with mitochondrial membranes

  • BioID or APEX2 fusion constructs to identify proximal proteins

Table 2: Subcellular Localization Methods Comparison

How can CRISPR-Cas9 technology be optimally designed to study C1orf98 function?

Designing CRISPR-Cas9 experiments for C1orf98 functional studies:

• Guide RNA design:

  • Design 3-4 sgRNAs targeting different exons of the C1orf98 gene

  • Focus on early exons to maximize disruption of protein function

  • Check for off-target effects using prediction algorithms

  • Consider the PAM site accessibility in the genomic context

• Knockout validation strategies:

  • Genomic validation: T7 endonuclease assay, Sanger sequencing of the targeted region

  • Transcript validation: RT-PCR and qPCR with primers spanning the targeted region

  • Protein validation: Western blotting with validated antibodies

• Phenotypic analysis pipeline:

  • Start with broad screens (viability, proliferation, morphology)

  • Progress to more specific assays based on bioinformatic predictions

  • Compare with phenotypes of related genes or pathways

• Rescue experiments:

  • Re-express wild-type C1orf98 to confirm specificity of observed phenotypes

  • Create domain mutants to identify functional regions

  • Use inducible systems to study temporal aspects of phenotypes

• Controls:

  • Non-targeting sgRNA controls

  • Knockout of genes with known phenotypes as positive controls

  • Isogenic cell lines differing only in C1orf98 status

These approaches follow true experimental design principles by manipulating independent variables (C1orf98 presence/function) while controlling for confounding factors .

What are the most effective approaches for identifying the molecular function of uncharacterized proteins like C1orf98?

To identify molecular functions of C1orf98:

• Integrated bioinformatics analysis:

  • Sequence-based predictions: conservation analysis, domain prediction, structural modeling

  • Expression correlation analysis: identification of genes with similar expression patterns

  • Network-based approaches: incorporation into protein-protein interaction networks

• Omics-based functional profiling:

  • Transcriptomics: RNA-seq after knockdown/overexpression to identify affected pathways

  • Proteomics: Global proteome changes and post-translational modification alterations

  • Metabolomics: Changes in metabolite profiles to infer biochemical influences

• Biochemical activity screening:

  • Enzymatic activity assays based on predicted domains

  • DNA/RNA binding assays if predicted to interact with nucleic acids

  • Lipid binding assays if predicted to interact with cellular membranes

• Genetic interaction mapping:

  • CRISPR screening to identify synthetic lethal or synthetic viable interactions

  • Double knockdown/knockout studies with predicted pathway components

  • Suppressor screens to identify genes that rescue C1orf98 loss phenotypes

This multi-faceted approach has proven successful for characterizing previously uncharacterized proteins like C17orf80, which was found to associate with mitochondrial membranes and nucleoids .

How can I design experiments to determine if C1orf98 interacts with nucleic acids or cellular membranes?

Given that some uncharacterized proteins like C17orf80 have been found to interact with nucleic acids (mitochondrial DNA) and membranes , similar investigations for C1orf98 are warranted:

• Nucleic acid interaction studies:

  • Electrophoretic mobility shift assays (EMSA) with various DNA/RNA substrates

  • Chromatin immunoprecipitation (ChIP) or RNA immunoprecipitation (RIP)

  • DNase/RNase treatment followed by co-immunoprecipitation to determine if interactions are nucleic acid-dependent

  • CLIP-seq or similar technologies to identify binding sites if RNA interaction is suspected

• Membrane association studies:

  • Membrane flotation assays to determine if C1orf98 associates with membranes

  • Protease protection assays to determine topology

  • Detergent resistance assays to characterize membrane microdomain association

  • FRAP (fluorescence recovery after photobleaching) to assess membrane dynamics

• Experimental design considerations:

  • Include appropriate controls (known DNA/RNA binding proteins, known membrane proteins)

  • Test multiple conditions (salt concentration, pH) to identify interaction requirements

  • Consider post-translational modifications that might regulate these interactions

These approaches should incorporate true experimental design principles by systematically manipulating variables and including appropriate controls .

What strategies can be employed to overcome expression and purification challenges with C1orf98?

Expression and purification of recombinant proteins, especially uncharacterized ones like C1orf98, often present technical challenges:

• Expression optimization:

  • Test multiple expression systems (bacterial, insect, mammalian)

  • For mammalian expression (as used for commercial C1orf98 ), optimize:

    • Cell line selection (HEK293, CHO, etc.)

    • Transfection conditions

    • Induction parameters

    • Harvest timing

• Solubility enhancement strategies:

  • Fusion tags: MBP, GST, SUMO, or TRX to increase solubility

  • Co-expression with chaperones

  • Expression at lower temperatures

  • Inclusion of specific additives in lysis buffer

• Purification optimization:

  • Multi-step purification strategy (e.g., affinity chromatography followed by size exclusion)

  • Screen different buffer conditions (pH, salt, additives)

  • Consider on-column refolding for proteins expressed in inclusion bodies

• Stability enhancement:

  • Addition of glycerol (5-50%) as recommended for C1orf98

  • Addition of reducing agents if cysteine residues are present

  • Use of protease inhibitors throughout purification

Table 3: Troubleshooting Guide for C1orf98 Expression and Purification

How can I design experiments to investigate potential post-translational modifications of C1orf98?

Post-translational modifications (PTMs) often regulate protein function and can be crucial for understanding uncharacterized proteins:

• Computational prediction:

  • Use algorithms to predict potential PTM sites (phosphorylation, glycosylation, ubiquitination)

  • Compare predictions across species to identify conserved modification sites

• Mass spectrometry-based approaches:

  • Enrichment strategies for specific PTMs:

    • Phosphorylation: TiO2 or IMAC enrichment

    • Ubiquitination: di-Gly antibody pulldown

    • Glycosylation: lectin affinity or hydrazide chemistry

  • Targeted and untargeted MS approaches

  • Quantitative comparison across different cellular conditions

• Site-directed mutagenesis validation:

  • Mutate predicted PTM sites to non-modifiable residues

  • Create phosphomimetic mutations (e.g., Ser to Asp/Glu)

  • Assess functional consequences through phenotypic assays

• PTM-specific antibodies:

  • When available, use modification-specific antibodies

  • Western blotting under different cellular conditions

  • Immunoprecipitation followed by mass spectrometry

These approaches follow experimental design principles by manipulating variables (cellular conditions, mutation of modification sites) and measuring outcomes (function, localization, interactions) .

What are the common technical challenges in studying uncharacterized proteins like C1orf98 and how can they be addressed?

Common technical challenges and their solutions:

• Antibody specificity issues:

  • Validate antibodies using knockout/knockdown controls

  • Use multiple antibodies targeting different epitopes

  • Complement antibody-based methods with tag-based approaches

• Expression level variability:

  • Use inducible expression systems to control levels

  • Quantify expression in each experiment

  • Normalize results to expression levels

• Functional redundancy masking phenotypes:

  • Identify and simultaneously target homologous proteins

  • Use different cell types or stress conditions to reveal phenotypes

  • Consider compensatory mechanisms in data interpretation

• Protein instability:

  • Optimize buffer conditions as described for C1orf98

  • Consider co-expression with stabilizing interaction partners

  • Use proteasome or lysosome inhibitors to assess degradation pathways

• Conflicting results between systems:

  • Systematically compare experimental conditions

  • Consider cell type-specific effects

  • Evaluate differences in protein levels or post-translational modifications

Addressing these challenges requires careful experimental design with appropriate controls and validation approaches following true experimental research design principles .

How should I validate the specificity of tools and reagents for C1orf98 research?

Validating research tools for C1orf98 studies:

• Antibody validation:

  • Western blot analysis with overexpression and knockdown/knockout controls

  • Immunoprecipitation followed by mass spectrometry

  • Multiple antibodies targeting different epitopes should yield consistent results

  • Peptide competition assays to confirm specificity

• Overexpression constructs:

  • Sequence verification

  • Expression level quantification

  • Assessment of tag interference with function or localization

  • Comparison of different tags and positions (N- or C-terminal)

• siRNA/sgRNA validation:

  • Quantification of knockdown efficiency at mRNA and protein levels

  • Use of multiple non-overlapping siRNAs/sgRNAs

  • Rescue experiments with expression constructs resistant to knockdown

  • Testing for off-target effects through transcriptome analysis

• Recombinant protein quality control:

  • Purity assessment (>85% for commercial C1orf98 )

  • Activity/functionality assays

  • Stability monitoring over time

  • Batch-to-batch consistency evaluation

Rigorous validation ensures that experimental outcomes reflect true biological phenomena rather than artifacts, aligning with principles of true experimental design .

What are the emerging trends and methodologies in uncharacterized protein research applicable to C1orf98?

Several emerging approaches are transforming uncharacterized protein research:

• Integrative structural biology:

  • Combining cryo-EM, X-ray crystallography, NMR, and computational modeling

  • High-throughput protein structure prediction using AI tools like AlphaFold

  • Structure-based function prediction

• Single-cell multi-omics:

  • Correlating protein levels with transcriptome and metabolome at single-cell resolution

  • Revealing cell type-specific functions

  • Identifying context-dependent interactions

• Genome-wide interaction mapping:

  • CRISPR screens for genetic interactions

  • Protein-protein interaction mapping in native contexts

  • Dynamic interactome analysis under different conditions

• Proximity proteomics advancements:

  • Similar to approaches used for C17orf80 characterization

  • Enhanced spatial resolution with split-BioID or TurboID systems

  • Temporal control with optogenetic or chemical induction

• In situ structural and functional analysis:

  • Super-resolution microscopy for protein localization

  • FRET-based interaction and conformation studies

  • Optogenetic tools for acute functional perturbation

These methodologies, when applied to C1orf98, could rapidly accelerate our understanding of its function and relevance to human biology and disease.

How can I design a comprehensive research program to fully characterize C1orf98?

A systematic research program for C1orf98 characterization would include:

• Phase 1: Foundational characterization (0-12 months)

  • Expression profiling across tissues and conditions

  • Subcellular localization

  • Interactome mapping

  • Initial loss-of-function phenotyping

• Phase 2: Mechanistic investigations (12-24 months)

  • Structure determination

  • Biochemical activity assays

  • Detailed phenotypic analysis in multiple cell types

  • PTM identification and functional relevance

• Phase 3: Physiological context (24-36 months)

  • Mouse models (knockout/knockin)

  • Tissue-specific functions

  • Disease relevance

  • Therapeutic potential assessment

This program should adhere to true experimental design principles, with careful control of variables, appropriate randomization, and rigorous statistical analysis .