C1orf109 Antibody

What is C1orf109 and what cellular functions does it perform?

C1orf109 is a 23 kDa protein (203 amino acids) encoded by the C1orf109 gene (Gene ID: 54955). It functions as a critical factor in ribosome biogenesis, specifically controlling a late step of human pre-60S maturation in the cytoplasm. Research has demonstrated that C1orf109 works together with three other proteins - SPATA5, SPATA5L1, and CINP - to promote the recycling of RSL24D1 from cytoplasmic pre-60S ribosomal subunits back to the nucleolus . Loss of C1orf109 impairs global protein synthesis and causes defects in ribosome maturation . While initially linked with cell proliferation, more recent studies have established its molecular function in the ribosome assembly pathway .

What are the validated applications for C1orf109 antibodies?

Based on current research literature and commercial antibody validation data, C1orf109 antibodies have been successfully employed in:

When designing experiments, researchers should consider that optimal dilutions may be sample-dependent and validation in your specific experimental system is recommended .

What is the specificity and reactivity profile of C1orf109 antibodies?

Commercial C1orf109 antibodies typically show reactivity with human samples . The Proteintech rabbit polyclonal antibody (25552-1-AP) has been specifically validated with K-562 cells, LNCaP cells, and U-251 cells . This antibody targets the full C1orf109 protein and has been produced using a C1orf109 fusion protein immunogen (Ag22147) .

Researchers should be aware that when investigating C1orf109's interactions with its partners (SPATA5, SPATA5L1, and CINP), the specificity of the antibody becomes especially important. Cross-reactivity testing is recommended if studying these interacting proteins simultaneously, particularly given the structural similarities between C1orf109 and CINP .

What are the recommended protocols for using C1orf109 antibodies in Western blotting?

For optimal Western blot results with C1orf109 antibodies, the following protocol is recommended:

• Sample Preparation:

  • Lyse cells in appropriate buffer containing protease inhibitors

  • The expected molecular weight of C1orf109 is 23 kDa

• Electrophoresis and Transfer:

  • Standard SDS-PAGE protocols are suitable

  • Semi-dry or wet transfer to PVDF or nitrocellulose membranes

• Antibody Incubation:

  • Block membrane with 5% non-fat milk or BSA in TBST

  • Incubate with primary C1orf109 antibody at 1:1000-1:6000 dilution

  • Wash and incubate with appropriate secondary antibody

• Controls:

  • Include lysate from cells known to express C1orf109 (e.g., K-562, LNCaP, or U-251 cells)

  • Consider using C1orf109 knockout/knockdown samples as negative controls

Storage recommendations: Store antibody at -20°C. For long-term storage, aliquoting is unnecessary .

How can C1orf109 antibodies be utilized to study ribosome biogenesis pathways?

C1orf109 antibodies provide powerful tools for investigating ribosome biogenesis through several complementary approaches:

• Co-immunoprecipitation studies: Use C1orf109 antibodies to pull down associated proteins and confirm interactions with SPATA5, SPATA5L1, and CINP. This approach has been used successfully with DSP crosslinking to demonstrate these interactions .

• Subcellular localization: Monitor the localization of C1orf109 and its partners during ribosome assembly using immunofluorescence microscopy. This has been valuable in showing how loss of C1orf109 affects the localization of ribosomal proteins such as RPL28 and RSL24D1 .

• Ribosome association studies: Combine polysome profiling with Western blotting using C1orf109 antibodies to determine which ribosomal fractions contain C1orf109. Research has shown that C1orf109 and its partners function in pre-60S maturation .

• Pulse-labeling experiments: Use antibodies to monitor the impact of C1orf109 knockdown/knockout on newly synthesized ribosomes, particularly in conjunction with SNAP-tag approaches or metabolic labeling as demonstrated in recent studies .

When designing these experiments, researchers should consider that C1orf109 may not directly interact with ribosomes under all conditions tested, though it clearly affects ribosome biogenesis through its partnerships with SPATA5, SPATA5L1, and CINP .

What methodologies are effective for studying C1orf109's interaction with its binding partners?

Multiple complementary approaches should be employed to comprehensively characterize C1orf109's interactions:

• Immunoprecipitation with crosslinking: DSP crosslinking has successfully demonstrated that C1orf109 interacts with SPATA5, SPATA5L1, and CINP . Protocol recommendations:

  • Use appropriate crosslinking concentration and duration

  • Include appropriate negative controls (IgG, knockout cells)

  • Validate interactions with reciprocal immunoprecipitations

• SNAP-tag pull-down assays: This approach has been effective for investigating the association of these proteins with ribosomes . The method involves:

  • Expression of SNAP-tagged ribosomal proteins (e.g., RPL28)

  • Pull-down with benzylguanine beads

  • Western blot analysis for C1orf109 and partner proteins

• Sucrose gradient fractionation: This technique can determine whether C1orf109 and its partners co-fractionate with specific ribosomal subunits . The approach requires:

  • Careful preparation of cytoplasmic lysates

  • Ultra-centrifugation through sucrose gradients

  • Analysis of fractions by Western blotting

• Rescue experiments: Overexpression of C1orf109 in knockout cells can confirm specificity and functional relationships. Research has shown C1orf109 overexpression rescues defects in C1orf109-knockout cells but not in CINP-knockout cells, suggesting non-redundant functions despite structural similarities .

How can researchers effectively validate C1orf109 knockout or knockdown models?

Rigorous validation of C1orf109 knockout/knockdown models is essential for experimental interpretation. Recommended approaches include:

• Western blot verification: Use C1orf109 antibodies to confirm protein depletion. Expected observations include:

  • Complete absence of the 23 kDa band in knockout models

  • Significant reduction in knockdown models

  • Potential changes in levels of interaction partners (loss of C1orf109 results in decreased levels of SPATA5, SPATA5L1, and CINP)

• Functional validation:

  • Ribosome maturation assays: Monitor nucleolar retention of RPL28 (can be labeled with SNAP-tag approaches)

  • RSL24D1 localization: Observe redistribution from nucleolus to cytoplasm

  • rRNA processing: Northern blot analysis should show increased unprocessed 47S/45S pre-rRNA and reduced 12S pre-rRNA

  • Translation efficiency: Measure using AHA pulse-labeling (expect reduced protein synthesis)

• Polysome profile analysis: Expect:

  • Imbalance between 40S and 60S subunits

  • Formation of half-mers (stalled 48S initiation complexes)

  • These are indicators of reduced functional 60S subunits

• Genotyping: Confirm genomic modifications by sequencing the targeted locus when using CRISPR-based approaches for knockout generation .

What are the challenges in detecting endogenous C1orf109 in different cell types and tissues?

Researchers face several challenges when detecting endogenous C1orf109:

• Expression level variations: C1orf109 expression may vary significantly between cell types. Validated detection has been demonstrated in:

  • K-562 cells (human myelogenous leukemia)

  • LNCaP cells (human prostate cancer)

  • U-251 cells (human glioblastoma)

  • HEK293T cells (used in ribosome biogenesis studies)

• Technical considerations:

  • Sample preparation: Complete lysis is critical; C1orf109's association with large complexes may affect extraction efficiency

  • Antibody sensitivity: Higher antibody concentrations may be needed for low-expressing samples

  • Signal amplification: Consider enhanced chemiluminescence systems for Western blot detection of low abundance proteins

• Distinguishing signal from background:

  • Knockout/knockdown controls are essential for validating specificity

  • C1orf109's interaction with SPATA5, SPATA5L1, and CINP may affect epitope accessibility

  • The protein's involvement in complexes may result in altered migration patterns

• Cross-reactivity concerns:

  • Given the structural similarity between C1orf109 and CINP, validation with multiple antibodies targeting different epitopes is advisable

How can C1orf109 antibodies be incorporated into ribosome assembly pathway studies?

Strategic integration of C1orf109 antibodies into ribosome assembly studies can provide valuable insights:

• Sequential immunoprecipitation strategy:

  • First IP: Pull down SNAP-tagged ribosomal proteins (e.g., RPL28-SNAP)

  • Second IP: Use C1orf109 antibodies on the eluate to identify associated proteins

  • Western blot for SPATA5, SPATA5L1, and CINP to map the interaction network

• Temporal analysis of ribosome maturation:

  • Pulse-chase experiments with SNAP-tagged RPL28

  • Time-course immunoprecipitation with C1orf109 antibodies

  • This approach can map when C1orf109 associates with maturing ribosomes

• Combining with rRNA processing analysis:

  • Northern blot analysis of rRNA processing using ITS1 and ITS2 probes

  • Western blot of the same samples with C1orf109 antibodies

  • Correlate C1orf109 levels with specific rRNA processing steps

• Targeted analysis of RSL24D1 recycling:

  • Immunoprecipitate C1orf109 and probe for RSL24D1

  • Alternatively, immunoprecipitate RSL24D1 and probe for C1orf109

  • This directly investigates the role of C1orf109 in RSL24D1 recycling from pre-60S subunits

These approaches benefit from the foundation established in published work showing C1orf109's role alongside SPATA5, SPATA5L1, and CINP in the late steps of human pre-60S ribosomal subunit maturation .

What experimental approaches can resolve conflicting data regarding C1orf109 function?

When faced with conflicting results regarding C1orf109 function, consider these methodological approaches:

• Cell type-specific analysis:

  • Compare C1orf109 function across multiple cell lines

  • Use the same antibody and standardized protocols

  • Cell-specific factors may influence C1orf109 behavior

• Domain-specific functional studies:

  • Generate truncation mutants of C1orf109

  • Perform rescue experiments with these constructs

  • Map which regions are essential for interaction with partners and ribosome maturation

• Quantitative assessment of protein complexes:

  • Size exclusion chromatography combined with Western blotting

  • Blue native PAGE to preserve protein complexes

  • Density gradient centrifugation to separate complexes by size

  • These approaches can determine if C1orf109 exists in multiple distinct complexes

• Addressing the relationship with CINP:

  • Despite structural similarities, C1orf109 and CINP appear to have non-redundant functions

  • Design experiments that distinguish between:

    • Direct effects of C1orf109 loss

    • Indirect effects through destabilization of the entire complex

    • CINP-specific functions versus C1orf109-specific functions

• Validate using complementary techniques:

  • If antibody-based methods give conflicting results, confirm with genetic tagging approaches

  • Combine with live-cell imaging of fluorescently tagged proteins

  • Use proximity ligation assays to verify protein-protein interactions in situ

What are the optimal methods for quantifying C1orf109 levels in experimental samples?

Accurate quantification of C1orf109 requires rigorous methodology:

• Western blot quantification:

  • Use standardized loading controls (e.g., GAPDH, β-actin)

  • Include a standard curve of recombinant C1orf109 for absolute quantification

  • Recommended dilution range: 1:1000-1:6000 for primary antibody

  • Apply digital image analysis with appropriate normalization

• ELISA-based quantification:

  • Commercial C1orf109 antibodies have been validated for ELISA applications

  • Develop a sandwich ELISA using antibodies recognizing different epitopes

  • Include standard curves with recombinant protein

• Mass spectrometry approaches:

  • Targeted MS methods such as selected reaction monitoring (SRM)

  • Use isotopically labeled peptide standards derived from C1orf109

  • This approach can be particularly valuable when studying post-translational modifications

• qPCR correlation:

  • While measuring mRNA rather than protein, qPCR can provide complementary data

  • Correlate transcript and protein levels to understand regulation

  • Particularly useful when antibody detection is challenging

When quantifying C1orf109, researchers should be aware that loss of C1orf109 affects levels of its binding partners (SPATA5, SPATA5L1, and CINP) , which may complicate interpretation of results in knockdown/knockout systems.

How can researchers effectively use C1orf109 antibodies in pre-60S ribosomal subunit maturation studies?

To leverage C1orf109 antibodies effectively in pre-60S maturation studies:

• Combined analysis of pre-rRNA processing and C1orf109 levels:

  • Northern blotting with ITS1/ITS2 probes to monitor pre-rRNA processing steps

  • Western blotting of the same samples for C1orf109

  • Expected pattern in C1orf109 dysfunction: increased 47S/45S pre-rRNA, reduced 12S pre-rRNA, decreased 28S:18S ratio

• Polysome profile analysis with C1orf109 immunoblotting:

  • Fractionate polysomes on sucrose gradients

  • Probe each fraction for C1orf109 and RSL24D1

  • In wild-type cells, RSL24D1 should fractionate with pre-60S but not with monosomes/polysomes

  • In C1orf109-deficient cells, expect RSL24D1 in 60S, monosome, and polysome fractions (except heaviest polysomes)

• Temporal analysis of ribosome maturation:

  • Use EU pulse-labeling to track nascent nucleolar/nuclear RNA

  • Combine with AHA pulse-labeling to assess translation

  • Correlate with C1orf109 levels by Western blotting

  • Loss of C1orf109 should result in reduced labeled nascent RNA and translation

• SNAP-tag pull-down strategy:

  • Generate cell lines expressing SNAP-tagged RPL28

  • Pull down SNAP-tagged ribosomes using benzylguanine beads

  • Western blot for C1orf109, SPATA5, SPATA5L1, CINP, and RSL24D1

  • This approach can determine the composition of maturing ribosomes

These methodologies directly build on published work establishing C1orf109's role in pre-60S ribosomal subunit maturation .

What are the key considerations for antibody storage and handling to maintain optimal performance?

Proper handling of C1orf109 antibodies is essential for experimental reproducibility:

• Storage recommendations:

  • Store at -20°C in the provided buffer (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

  • According to manufacturer data, the antibody is stable for one year after shipment

  • For common antibody formats, aliquoting is unnecessary for -20°C storage

• Handling guidelines:

  • Avoid repeated freeze-thaw cycles

  • If using small volumes (e.g., 20μl), products may contain 0.1% BSA for stability

  • Return to -20°C promptly after use

  • Work with antibodies on ice when possible

• Quality control considerations:

  • Perform periodic validation against positive control samples (K-562, LNCaP, or U-251 cells)

  • Compare new lots with previously validated lots

  • Consider including fresh positive controls with each experiment

• Dilution practices:

  • Prepare working dilutions freshly before use

  • For Western blotting, recommended dilutions range from 1:1000-1:6000

  • Optimize dilutions empirically for each application and sample type

Following these guidelines will help maintain antibody performance throughout your research project.

How can researchers troubleshoot weak or non-specific signals when using C1orf109 antibodies?

When encountering detection problems with C1orf109 antibodies, consider these methodical troubleshooting approaches:

• Weak signal solutions:

  • Increase antibody concentration (within recommended range of 1:1000-1:6000)

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

  • Enhance protein extraction using stronger lysis buffers

  • Use more sensitive detection systems (enhanced chemiluminescence)

  • Confirm C1orf109 expression in your sample (compare to K-562, LNCaP, or U-251 cells)

• Non-specific binding remedies:

  • Optimize blocking conditions (try 5% non-fat milk vs. BSA)

  • Increase washing duration and volume

  • Use higher dilution of antibody (closer to 1:6000)

  • Pre-adsorb antibody with lysate from C1orf109 knockout cells

  • Validate specificity with knockout/knockdown controls

• Background reduction:

  • Use fresher membranes and reagents

  • Ensure complete removal of SDS from transfer buffer

  • Consider alternative secondary antibodies

  • Increase Tween-20 concentration in wash buffer slightly

• Experimental controls:

  • Include positive control lysates (K-562, LNCaP, or U-251 cells)

  • Use siRNA knockdown or CRISPR knockout samples as negative controls

  • Consider peptide competition assays to confirm specificity

If problems persist, epitope masking due to protein-protein interactions may be occurring, particularly given C1orf109's association with SPATA5, SPATA5L1, and CINP .

What are the recommended approaches for optimizing C1orf109 immunoprecipitation experiments?

Successful immunoprecipitation of C1orf109 requires careful optimization:

• Lysis buffer optimization:

  • Test different lysis buffers (RIPA, NP-40, Triton X-100)

  • Include appropriate protease inhibitors

  • Consider phosphatase inhibitors if studying phosphorylation states

  • For capturing transient interactions, include crosslinkers like DSP as demonstrated in published work

• Antibody binding strategies:

  • Pre-bind antibody to beads (Protein A/G) before adding lysate

  • Alternatively, form antibody-antigen complexes in solution before adding beads

  • Optimize antibody amount (typically 1-5 μg per mg of total protein)

  • Consider crosslinking antibody to beads to prevent co-elution

• Washing optimization:

  • Begin with manufacturer's recommended wash buffer

  • Test wash stringency by modifying salt concentration (150-500 mM NaCl)

  • Adjust detergent concentrations to balance specific binding versus background

  • Multiple short washes often perform better than fewer long washes

• Elution strategies:

  • For Western blot analysis: denaturing elution with SDS sample buffer

  • For maintaining protein complexes: native elution with excess peptide

  • For mass spectrometry: on-bead digestion may reduce contamination

• Controls:

  • Include IgG control from same species as C1orf109 antibody

  • Use lysate from C1orf109 knockout cells as negative control

  • Consider including RNase treatment to distinguish RNA-dependent interactions

When investigating C1orf109 interactions with SPATA5, SPATA5L1, and CINP, DSP crosslinking has proven effective in capturing these associations .

What considerations should researchers make when selecting between different commercial C1orf109 antibodies?

Strategic selection of C1orf109 antibodies should consider:

• Epitope targeting:

  • Antibodies targeting different regions may perform differently in specific applications

  • N-terminal antibodies may be preferable when studying C-terminal interactions

  • C-terminal antibodies may be useful for distinguishing C1orf109 from CINP, which has structural similarities

• Host species and format:

  • Rabbit polyclonal antibodies are commonly available and validated

  • Consider host species compatibility with other antibodies in multiplexed applications

  • Monoclonal antibodies may offer greater batch-to-batch consistency

• Validation extent:

  • Review validation data in applications of interest

  • Confirmed applications include Western blot and ELISA

  • Check if validation includes knockout/knockdown controls

  • Verify reactivity with human samples and specific cell lines (K-562, LNCaP, U-251)

• Application-specific performance:

  • For Western blotting: antibodies validated at 1:1000-1:6000 dilution

  • For immunoprecipitation: consider antibodies specifically validated for this application

  • For immunofluorescence: evaluate subcellular localization data if available

• Cross-reactivity:

  • Given structural similarities between C1orf109 and CINP , assess cross-reactivity data

  • Consider testing in CINP knockout cells to confirm specificity

Researchers should validate antibody performance in their specific experimental system regardless of manufacturer claims.

What emerging research directions are anticipated for C1orf109 studies?

Based on current knowledge about C1orf109, several promising research directions are emerging:

• Structural biology approaches:

  • Building on AlphaFold predictions , determine experimental structures of C1orf109 alone and in complex with partners

  • Investigate whether C1orf109 and CINP can form heterodimers or hetero-oligomers

  • Resolve the structural basis for specificity between C1orf109 and CINP despite their similar predicted structures

• Mechanistic studies of ribosome biogenesis:

  • Determine precisely how C1orf109 contributes to RSL24D1 recycling

  • Investigate whether C1orf109 regulates the ATPase activity of SPATA5 and SPATA5L1

  • Characterize how these four proteins (C1orf109, SPATA5, SPATA5L1, and CINP) coordinate to perform functions carried out by a single protein (Drg1) in yeast

• Tissue-specific and developmental regulation:

  • Examine expression patterns of C1orf109 across different tissues and developmental stages

  • Investigate potential connections to SPATA5-associated neurodevelopmental disorders

  • Determine if C1orf109 dysfunction contributes to human disease pathology

• Therapeutic potential:

  • Explore whether targeting the C1orf109 pathway might provide therapeutic opportunities in diseases with dysregulated ribosome biogenesis

  • Investigate connections between C1orf109 and cancer, given its links to cell proliferation

These research directions will benefit from continued development and application of specific antibodies against C1orf109 and its interaction partners.

How can researchers effectively integrate C1orf109 antibody data with other experimental approaches?

Maximizing research impact requires integrating antibody-based data with complementary methodologies:

• Multi-omics integration:

  • Correlate proteomic data on C1orf109 levels/interactions with transcriptomic data

  • Integrate ribosome profiling data to assess translation effects

  • Combine with structural biology approaches (cryo-EM of ribosome intermediates)

• Genetic and antibody-based approaches:

  • Compare CRISPR knockout phenotypes with antibody neutralization experiments

  • Use antibodies to validate genetic screening hits related to ribosome biogenesis

  • Combine with gene expression analyses to identify regulatory networks

• Live-cell and fixed-cell techniques:

  • Correlate antibody-based fixed-cell immunofluorescence with live-cell imaging using fluorescently tagged C1orf109

  • Use proximity ligation assays to verify protein-protein interactions in situ

  • Validate binding partners identified in IP-MS experiments with co-localization studies

• Evolutionary analyses:

  • Compare antibody reactivity across species to complement evolutionary analyses

  • Investigate whether the split of Drg1's function into multiple proteins (C1orf109, SPATA5, SPATA5L1, and CINP) in humans represents specialization or redundancy

  • Correlate structural predictions with antibody epitope accessibility across species