c1d Antibody

What is C1D protein and what are its primary biological functions?

C1D (Nuclear nucleic acid-binding protein C1D) is a ubiquitously expressed nuclear protein with a calculated molecular weight of 16 kDa that functions as a nuclear receptor co-repressor. This protein plays multiple critical roles in cellular processes, including:

• Recruitment of the RNA exosome complex to pre-rRNA to mediate the 3'-5' end processing of the 5.8S rRNA (potentially involving MPHOSPH6)

• Activation of PRKDC (DNA-PK) in the presence of both linear and supercoiled DNA

• Induction of apoptosis in a p53/TP53-dependent manner

• Regulation of the TRAX/TSN complex formation

• Potentiation of transcriptional repression by nuclear receptors NR1D1 and THRB

C1D was initially identified by screening a cDNA expression library with monoclonal antibodies raised against residual polypeptides that remain attached to DNA following aggressive purification methods including SDS, proteinase K, and phenol extraction .

What applications are validated for C1D antibodies and what are their recommended dilutions?

C1D antibodies have been validated for multiple applications with specific recommended dilutions:

It is strongly recommended that researchers titrate these antibodies in each testing system to obtain optimal results, as performance can be sample-dependent .

Which sample types and cell lines have been validated for C1D antibody detection?

The following sample types have been validated for C1D antibody detection:

Cell lines:

• DU 145 cells (prostate cancer) - Positive in WB and IP

• HeLa cells (cervical adenocarcinoma)

• HT-1080 cells (human fibrosarcoma)

• MDA-MB-231 cells (breast adenocarcinoma)

Tissue samples:

• Human prostate cancer tissue - Positive in IHC

• Human heart tissue and fetal heart tissue

• Human skeletal muscle tissue

• Human thyroid tissue

• Human fetal kidney tissue

These validated samples provide excellent reference points when establishing C1D antibody protocols in new experimental systems.

How does C1D interact with DNA-PK and what is the significance of this interaction in DNA repair?

C1D interacts specifically with the putative leucine zipper (LZ) region of DNA-PK catalytic subunit (DNA-PKcs). Through yeast two-hybrid screens and in vitro binding assays, researchers have demonstrated that:

• C1D interacts directly with the leucine zipper motif of DNA-PKcs

• This interaction is direct and not mediated by DNA, as confirmed by ethidium bromide treatment experiments

• C1D does not interact directly with Ku, though these proteins can associate indirectly through DNA

• DNA-PKcs can simultaneously contact both C1D and Ku

The significance of this interaction lies in C1D's ability to activate DNA-PK not only in the presence of linear DNA (containing double-strand breaks) but also in the presence of supercoiled DNA. This suggests a novel mechanism for DNA-PK activation in vivo that may be independent of DNA double-strand breaks, potentially expanding our understanding of DNA-PK functions in cellular processes beyond classical DNA repair pathways .

Specific point mutations in the leucine zipper motif of DNA-PKcs abolish this interaction, confirming the specificity of the C1D-DNA-PKcs binding interface .

What methods can be used to study C1D's role in RNA processing using C1D antibodies?

C1D plays a role in recruiting the RNA exosome complex to pre-rRNA to mediate the 3'-5' end processing of the 5.8S rRNA . To investigate this function, researchers can employ the following methodological approaches using C1D antibodies:

• RNA Immunoprecipitation (RIP): Use C1D antibodies to precipitate RNA-protein complexes, followed by RT-PCR or sequencing to identify associated RNAs.

  Protocol outline:

  • Crosslink cells to preserve RNA-protein interactions

  • Lyse cells in non-denaturing conditions

  • Immunoprecipitate with C1D antibody (0.5-4.0 μg per reaction)

  • Extract and analyze associated RNAs

• Co-immunoprecipitation: Isolate C1D-containing complexes and analyze for the presence of RNA exosome components like EXOSC10, RRP6, or MPHOSPH6.

• Immunofluorescence co-localization: Use dual labeling with C1D antibodies and markers of nucleolar compartments to visualize co-localization with rRNA processing sites.

• Chromatin Immunoprecipitation (ChIP): Examine C1D association with rDNA loci to assess its role in co-transcriptional rRNA processing.

• Proximity Ligation Assay (PLA): Combine C1D antibodies with antibodies against RNA exosome components to visualize and quantify direct interactions in situ.

These approaches can provide mechanistic insights into how C1D contributes to RNA processing pathways and potentially identify new RNA targets or protein interactions.

How can researchers design appropriate controls for C1D antibody validation in experimental systems?

When validating C1D antibodies for experimental use, researchers should implement the following comprehensive control strategy:

Positive controls:

• Recombinant C1D protein or C1D-overexpressing cells

• Cell lines with confirmed C1D expression (e.g., DU 145, HeLa, HT-1080)

• Tissue samples with known C1D expression (e.g., human heart, skeletal muscle)

Negative controls:

• Primary antibody omission control

• Isotype control (rabbit IgG at the same concentration as the C1D antibody)

• Competitive peptide blocking (pre-incubating the antibody with excess immunizing peptide)

• C1D knockdown/knockout cells (if available)

Additional validation approaches:

• Testing multiple antibodies targeting different C1D epitopes

• Correlation of protein detection with C1D mRNA expression data

• Verification of the expected molecular weight (16 kDa) by Western blot

• Confirmation of anticipated subcellular localization (nuclear)

• Specificity validation across species if performing cross-species analysis

Implementation of these controls ensures the specificity and reliability of experimental results using C1D antibodies, and helps troubleshoot any inconsistent findings.

What is known about C1D's role in apoptosis and how can this be studied using C1D antibodies?

C1D can induce apoptosis in a p53/TP53-dependent manner , suggesting its involvement in cellular responses to genotoxic stress and regulation of cell death pathways. To investigate this function, researchers can employ the following methodological approaches:

• Immunoblotting time-course: Monitor C1D expression levels during apoptosis induction using validated dilutions (1:200-1:1000) and correlate with established apoptotic markers.

• Co-immunoprecipitation studies: Use C1D antibodies (0.5-4.0 μg per 1-3 mg lysate) to identify interactions with:

  • p53 and MDM2 (key regulators of apoptosis)

  • Bcl-2 family proteins

  • Caspases and their regulators

• Chromatin Immunoprecipitation (ChIP): Examine C1D binding to promoters of p53-regulated genes involved in apoptosis.

• Subcellular fractionation and immunoblotting: Track C1D translocation between nuclear and cytoplasmic compartments during apoptosis.

• Functional assays with antibody intervention: Use C1D antibodies to neutralize or deplete C1D in cellular systems, then measure effects on:

  • DNA fragmentation (TUNEL assay)

  • Phosphatidylserine externalization (Annexin V staining)

  • Caspase activation

  • Mitochondrial membrane potential

These approaches can elucidate the mechanisms by which C1D contributes to apoptotic pathways and potentially identify new therapeutic targets for diseases characterized by dysregulated apoptosis.

What are the optimal antigen retrieval methods for immunohistochemistry with C1D antibodies?

For optimal immunohistochemical detection of C1D in tissue samples, the following antigen retrieval methods are recommended:

Primary recommendation:

• Antigen retrieval with TE buffer pH 9.0

Alternative method:

• Antigen retrieval with citrate buffer pH 6.0

Detailed methodological protocol:

• Deparaffinize tissue sections completely in xylene (3 changes, 5 minutes each)

• Rehydrate through graded alcohols to distilled water

• Perform heat-induced epitope retrieval using one of the following methods:
  a. TE buffer (10mM Tris, 1mM EDTA, pH 9.0) at 95-98°C for 15-20 minutes
  b. Citrate buffer (10mM Sodium Citrate, pH 6.0) at 95-98°C for 15-20 minutes

• Allow slides to cool in the buffer for 20 minutes at room temperature

• Wash in PBS or TBS (3 changes, 5 minutes each)

• Proceed with blocking steps and antibody incubation using recommended dilutions (1:50-1:500)

Researchers should empirically determine the optimal retrieval method for their specific tissue samples, as different fixation methods and tissue types may respond differently to these protocols.

What is the recommended protocol for using C1D antibodies in immunoprecipitation studies?

Based on validated applications, the following protocol is recommended for immunoprecipitation using C1D antibodies:

Materials required:

• C1D antibody (recommended amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate)

• Protein A/G magnetic or agarose beads

• Cell lysis buffer (non-denaturing)

• Wash buffers

• Protease and phosphatase inhibitors

• SDS-PAGE sample buffer

Detailed protocol:

• Cell lysis:

  • Harvest cells (e.g., DU 145, which has been validated)

  • Lyse in non-denaturing buffer containing protease/phosphatase inhibitors

  • Clarify lysate by centrifugation (14,000 × g, 10 minutes, 4°C)

• Pre-clearing:

  • Incubate lysate with Protein A/G beads (40 μl of 50% slurry per ml) for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add C1D antibody to pre-cleared lysate (0.5-4.0 μg per 1-3 mg protein)

  • Incubate overnight at 4°C with gentle rotation

  • Add 40 μl Protein A/G beads and incubate for 2-4 hours at 4°C

• Washing:

  • Collect beads by centrifugation or magnetic separation

  • Wash 4-5 times with cold wash buffer

  • For DNA-dependent interactions, include ethidium bromide (50 μg/ml) in one wash to distinguish direct from DNA-mediated interactions

• Elution and analysis:

  • Resuspend beads in SDS-PAGE sample buffer

  • Heat at 95°C for 5 minutes

  • Analyze by SDS-PAGE and Western blotting

This protocol has been validated for detecting C1D interactions, including its association with DNA-PKcs .

How should researchers interpret C1D expression patterns in cancer versus normal tissues?

When analyzing differential C1D expression between normal and cancer tissues, researchers should consider the following analytical framework:

Methodological considerations:

• Standardization: Use identical processing, staining protocols, and antibody concentrations for all samples

• Quantification: Employ digital image analysis rather than subjective scoring when possible

• Cell-type specificity: Evaluate expression in specific cell populations rather than whole tissue

• Subcellular localization: Assess both intensity and localization patterns (nuclear vs. cytoplasmic)

Interpretative framework:

• Functional context: Interpret findings in the context of C1D's known roles in:

  • DNA repair and genomic stability

  • RNA processing

  • Apoptosis regulation

  • Transcriptional repression

• Correlation analysis: Examine correlations between C1D expression and:

  • Clinical parameters (stage, grade, survival)

  • DNA damage markers (γH2AX, 53BP1)

  • Apoptotic indices

  • RNA processing defects

• Comparative analysis: Consider tissue-specific baseline expression (e.g., C1D is detected in normal heart, skeletal muscle, and thyroid tissues)

• Validation: Confirm IHC findings with complementary methods such as Western blotting or qRT-PCR

C1D antibodies have been validated for detection in human prostate cancer tissue , providing a starting point for such comparative analyses. Given C1D's roles in fundamental cellular processes, changes in its expression may have significant implications for cancer biology and potential therapeutic approaches.

What might cause multiple bands in Western blots using C1D antibodies and how can this be resolved?

While C1D has a calculated molecular weight of 16 kDa , multiple bands might be observed in Western blotting. Here's a systematic approach to troubleshoot and resolve this issue:

Potential causes and solutions:

• Post-translational modifications:

  • Cause: Phosphorylation, ubiquitination, or SUMOylation of C1D

  • Solution: Use phosphatase treatment or specific inhibitors to confirm modification status

• Protein isoforms:

  • Cause: Alternative splicing of the C1D gene

  • Solution: Compare with recombinant C1D protein standards; validate with RT-PCR for splice variants

• Proteolytic degradation:

  • Cause: Sample preparation issues

  • Solution: Use fresh samples; enhance protease inhibitor cocktail; maintain samples at 4°C; avoid repeated freeze-thaw cycles

• Cross-reactivity:

  • Cause: Antibody binding to structurally similar proteins

  • Solution: Perform peptide competition assay; use alternative C1D antibody targeting different epitope; validate in C1D-depleted samples

• Incomplete denaturation:

  • Cause: C1D remaining in protein complexes

  • Solution: Increase SDS concentration, extend boiling time, add reducing agents

Optimization protocol:

• Prepare fresh lysates with comprehensive protease inhibitor cocktail

• Standardize protein loading (15-20 μg per lane)

• Test multiple antibody dilutions within the recommended range (1:200-1:1000)

• Include positive control (e.g., lysate from DU 145 cells)

• Compare results using different C1D antibodies if available

This systematic approach should help resolve multiple band issues and ensure specific detection of C1D protein.

How can researchers minimize background staining in immunohistochemistry with C1D antibodies?

Background staining in C1D immunohistochemistry can significantly impact result interpretation. Here's a comprehensive approach to minimize background:

Optimization protocol:

• Antibody dilution optimization:

  • Perform dilution series within recommended range (1:50-1:500)

  • Include positive and negative controls with each dilution

  • Select optimal dilution based on signal-to-noise ratio

• Antigen retrieval optimization:

  • Compare both recommended methods:
    a. TE buffer pH 9.0 (primary recommendation)
    b. Citrate buffer pH 6.0 (alternative method)

  • Test multiple retrieval durations (10, 15, 20 minutes)

• Enhanced blocking protocol:

  • Block endogenous peroxidase (3% H₂O₂, 10 minutes)

  • Use species-appropriate serum block (5-10%)

  • Include protein block (1-2% BSA or casein)

  • For tissues with high biotin, add avidin/biotin blocking step

  • Consider adding 0.1-0.3% Triton X-100 for enhanced antibody penetration

• Optimized washing:

  • Extend wash times (3 × 10 minutes instead of standard 3 × 5)

  • Use 0.05-0.1% Tween-20 in wash buffer

  • Ensure complete buffer exchanges between steps

• Secondary antibody optimization:

  • Use highly cross-adsorbed secondary antibodies

  • Titrate secondary antibody concentrations

  • Consider polymer detection systems for enhanced specificity

• Additional considerations:

  • Use freshly cut tissue sections

  • Reduce primary antibody incubation temperature (4°C overnight)

  • Apply hydrophobic barrier around tissue sections

  • Use automated staining platforms if available

Implementation of these systematic optimization steps should significantly reduce background staining while preserving specific C1D signal in immunohistochemical applications.

What are the key considerations when using C1D antibodies for co-immunoprecipitation of protein complexes?

Co-immunoprecipitation (co-IP) using C1D antibodies presents unique challenges due to C1D's interactions with DNA and its involvement in multiple protein complexes. Here are key methodological considerations:

Critical protocol elements:

• DNA-mediated interaction discrimination:

  • C1D binds DNA, potentially leading to false-positive interactions

  • Solution: Include ethidium bromide (50 μg/ml) during immunoprecipitation to disrupt protein-DNA interactions

  • Validation approach: Compare complex composition with and without ethidium bromide treatment

• Protein complex preservation:

  • Lysis conditions: Use gentle, non-denaturing buffers (e.g., NETN: 100mM NaCl, 1mM EDTA, 20mM Tris-HCl pH 8.0, 0.5% NP-40)

  • Temperature control: Maintain samples at 4°C throughout procedure

  • Crosslinking option: For transient interactions, consider reversible crosslinking (DSP or formaldehyde)

• Antibody optimization:

  • Amount: Use recommended 0.5-4.0 μg antibody per 1.0-3.0 mg protein

  • Incubation: Extend to overnight at 4°C for complete antigen capture

  • Orientation options: Consider using pre-bound antibody to beads or sequential addition

• Controls for validation:

  • Negative control: Isotype-matched IgG precipitation

  • Reciprocal IP: Confirm interactions by IP with antibodies against suspected partners

  • Input control: Analyze 5-10% of pre-IP lysate

  • Antibody-only control: Control for antibody heavy/light chain detection

• Analytical considerations:

  • Washing stringency: Balance between removing non-specific interactions and preserving genuine ones

  • Elution options: Consider native elution with peptide competition for functional studies

  • Detection method: Use clean detection antibodies (not cross-reactive with IP antibody)

These methodological refinements address the specific challenges of studying C1D interactions, particularly its unique ability to activate DNA-PK through both DNA-dependent and DNA-independent mechanisms .

How can C1D antibodies be utilized to investigate DNA repair mechanisms?

C1D's interaction with DNA-PK and its role in activating this kinase in both linear and supercoiled DNA contexts makes it an intriguing target for DNA repair research. Here are advanced methodological approaches using C1D antibodies:

• ChIP-sequencing for DNA damage response:

  • Method: Use C1D antibodies for chromatin immunoprecipitation followed by next-generation sequencing

  • Application: Map genomic binding sites of C1D before and after DNA damage

  • Protocol elements:

    • Crosslink cells with 1% formaldehyde (10 minutes, room temperature)

    • Sonicate chromatin to 200-500 bp fragments

    • Immunoprecipitate with validated C1D antibody

    • Prepare sequencing libraries from precipitated DNA

    • Analyze enrichment patterns at damage-prone regions

• Proximity ligation assay (PLA) for repair complex visualization:

  • Method: Combine C1D antibodies with antibodies against DNA repair factors

  • Application: Visualize and quantify in situ interactions at sites of DNA damage

  • Protocol elements:

    • Treat cells with DNA-damaging agents (e.g., ionizing radiation, etoposide)

    • Fix and permeabilize cells

    • Incubate with C1D antibody and antibody against potential partner (e.g., DNA-PKcs)

    • Apply PLA probes and perform rolling circle amplification

    • Quantify interaction foci in relation to damage markers

• In vitro kinase assays:

  • Method: Immunoprecipitate C1D-associated complexes and assess DNA-PK activity

  • Application: Determine how C1D regulates DNA-PK under different DNA structural contexts

  • Protocol elements:

    • Immunoprecipitate with C1D antibody using recommended amounts

    • Add recombinant substrate and γ-³²P-ATP

    • Compare kinase activity with linear versus supercoiled DNA templates

    • Analyze substrate phosphorylation by autoradiography or phospho-specific antibodies

• CRISPR-Cas9 C1D knockout phenotyping:

  • Method: Generate C1D knockout cell lines and assess DNA repair efficiency

  • Application: Determine functional significance of C1D in different repair pathways

  • Validation approach: Confirm knockout by Western blot with C1D antibodies

These approaches leverage C1D antibodies to explore its unique functions in DNA repair, particularly its ability to activate DNA-PK in contexts beyond classical double-strand breaks .

How can researchers use C1D antibodies to investigate auto-antibody responses in cancer patients?

Given C1D's nuclear localization and its presence in prostate cancer tissue , it may serve as a target for auto-antibody responses in cancer patients. Here's a methodological framework for such investigations:

• Serological screening for anti-C1D auto-antibodies:

  • Method: Develop ELISA using purified recombinant C1D protein

  • Application: Screen serum samples from cancer patients versus healthy controls

  • Protocol elements:

    • Coat plates with recombinant C1D protein

    • Incubate with patient sera at multiple dilutions

    • Detect bound human antibodies with anti-human IgG-HRP

    • Establish cutoff values based on healthy control populations

    • Validate positive results with Western blot confirmation

• Auto-antibody characterization:

  • Method: Epitope mapping using peptide arrays

  • Application: Identify immunodominant regions of C1D

  • Analytical approach: Compare epitope patterns between cancer types and correlation with disease stage

• Clinical correlation studies:

  • Method: Prospective analysis of anti-C1D auto-antibodies in patient cohorts

  • Application: Assess potential as diagnostic or prognostic biomarkers

  • Validation approach: Compare with established cancer biomarkers

• Multi-marker auto-antibody panels:

  • Method: Combine C1D with other nuclear antigens in multiplexed assays

  • Application: Enhance sensitivity and specificity for early cancer detection

  • Examples: Consider including established auto-antibody targets from validated panels like EarlyCDT-Lung (p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1, SOX2)

This research direction aligns with emerging evidence on the utility of auto-antibody panels for early cancer detection, as referenced in the context of lung cancer screening where auto-antibody panels demonstrated 91-93% specificity .

What cutting-edge approaches use C1D antibodies to study RNA exosome recruitment and function?

C1D's role in RNA exosome complex recruitment for 5.8S rRNA processing represents an important area for advanced investigation. Here are sophisticated methodological approaches using C1D antibodies:

• RIP-sequencing (RNA immunoprecipitation-sequencing):

  • Method: Immunoprecipitate C1D-bound RNAs followed by high-throughput sequencing

  • Application: Identify the complete repertoire of RNAs associated with C1D

  • Protocol elements:

    • Crosslink cells with UV or formaldehyde

    • Lyse cells and fragment RNA to 200-300 nt

    • Immunoprecipitate with C1D antibody

    • Extract and sequence associated RNAs

    • Compare with RNA exosome component IP profiles

• Proximity-dependent biotinylation (BioID or TurboID):

  • Method: Express C1D fused to biotin ligase, then capture biotinylated proteins

  • Application: Identify proteins in close proximity to C1D in living cells

  • Validation approach: Confirm interactions by co-IP with C1D antibodies

  • Output: Dynamic protein interaction network around C1D in different cellular compartments

• Single-molecule RNA visualization:

  • Method: Combine C1D immunofluorescence with RNA FISH

  • Application: Visualize co-localization of C1D with specific RNA targets

  • Analytical approach: Quantify co-localization at nucleolar and nuclear sites

• Exosome complex reconstitution:

  • Method: In vitro assembly of RNA exosome components with recombinant C1D

  • Application: Determine the biochemical requirements for C1D-mediated exosome activity

  • Validation approach: Use C1D antibodies to deplete C1D from cell extracts and test for complementation with recombinant protein

• In vitro RNA processing assays:

  • Method: Immunoprecipitate C1D-containing complexes and test RNA processing activity

  • Application: Directly measure the contribution of C1D to exosome function

  • Protocol elements:

    • Immunoprecipitate with C1D antibody using validated amounts

    • Add labeled RNA substrates

    • Analyze processing products by denaturing PAGE

    • Compare activity with wild-type versus mutant substrates

These approaches leverage C1D antibodies to provide mechanistic insights into how this protein contributes to RNA quality control and processing through exosome recruitment and activation.