C12orf60 Antibody

What is C12orf60 and why is it important for research?

C12orf60 (Chromosome 12 Open Reading Frame 60) is an uncharacterized protein that is believed to function as a potential regulator of cell proliferation and apoptosis. Its involvement in fundamental biological processes such as cell cycle regulation and programmed cell death makes it a promising target for investigation in cancer research and other disease studies. Understanding the function of C12orf60 could provide valuable insights into disease mechanisms and potentially lead to the development of novel therapeutic strategies . The protein's relatively unexplored nature makes it particularly interesting for researchers exploring new pathways in cell biology.

What are the validated applications for C12orf60 antibodies?

C12orf60 antibodies have been validated for multiple research applications, with varying recommended dilutions for optimal results:

These applications enable researchers to detect and analyze C12orf60 protein expression and localization in various cell types and tissue samples. When designing experiments, it's essential to validate the optimal dilution for your specific sample type and experimental conditions .

What is the optimal storage method for C12orf60 antibodies?

C12orf60 antibodies are typically available in liquid form containing preservatives like 0.03% Proclin 300 and 50% glycerol in PBS buffer (pH 7.4) . For long-term stability and maintenance of antibody activity:

• Store the antibody at -20°C or -80°C according to manufacturer recommendations

• Avoid repeated freeze-thaw cycles which can compromise antibody functionality

• If provided in lyophilized form, reconstitute in 100 μl of sterile distilled H₂O with 50% glycerol

• After reconstitution, the concentration is typically 1 mg/ml

Proper storage is critical for maintaining antibody specificity and sensitivity in experimental applications, especially for quantitative analyses like ELISA or Western blotting .

What sample types have been validated for C12orf60 antibody reactivity?

C12orf60 antibodies have been primarily validated for human samples, with demonstrated reactivity in:

• Human kidney tissue (using paraffin-embedded sections for IHC)

• Human pancreatic tissue (using paraffin-embedded sections for IHC)

• A549 cells (human lung carcinoma cell line for IF)

Some manufacturers predict cross-reactivity with mouse and rat C12orf60 based on sequence homology, though this requires empirical validation by the researcher . When working with non-human samples, preliminary testing at multiple antibody dilutions is recommended to confirm specificity before proceeding with full-scale experiments.

How can I optimize Western blotting protocols for C12orf60 detection?

For optimal Western blot results when detecting C12orf60:

• Expected molecular weight: The observed molecular weight of C12orf60 is approximately 27 kDa

• Sample preparation: Use RIPA buffer with protease inhibitors for cell/tissue lysis

• Loading control: Include β-actin or GAPDH as housekeeping proteins

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation: Use dilutions between 1:500-1:2000 in blocking solution overnight at 4°C

• Secondary antibody: Anti-rabbit IgG HRP-conjugated at 1:5000-1:10000 dilution

• Development: Use enhanced chemiluminescence (ECL) detection systems

• Optimization: If background is high, increase washing steps or adjust antibody concentrations

For quantitative analysis, ensure linear detection range using a dilution series of your sample and capture images before signal saturation occurs. This is particularly important when comparing C12orf60 expression levels across different experimental conditions or cell types.

What controls should be included in experiments using C12orf60 antibodies?

Robust experimental design for C12orf60 antibody applications should include the following controls:

• Positive control: Cell lines or tissues known to express C12orf60 (e.g., A549 cells have been validated)

• Negative control:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • Tissues/cells where C12orf60 expression is minimal or absent

• Specificity controls:

  • Preabsorption with immunogen peptide (Recombinant Human Uncharacterized protein C12orf60)

  • siRNA knockdown of C12orf60 (if available)

• Technical controls:

  • For IHC/IF: Secondary antibody-only controls to assess non-specific binding

  • For WB: Loading controls (β-actin, GAPDH, etc.)

  • For ELISA: Standard curve using recombinant C12orf60 protein

These controls help distinguish between specific antibody binding to C12orf60 and potential artifacts or non-specific signals, which is critical for publication-quality research and data interpretation.

What alternative approaches can I use to validate C12orf60 antibody results?

To strengthen confidence in C12orf60 antibody results, consider these complementary validation approaches:

• Orthogonal validation:

  • Correlate protein detection with mRNA expression (RT-qPCR)

  • Use multiple antibodies targeting different epitopes of C12orf60

  • Employ mass spectrometry-based protein identification

• Functional validation:

  • Compare results before and after gene silencing (siRNA, CRISPR/Cas9)

  • Overexpression studies with tagged versions of C12orf60

• Alternative detection methods:

  • Consider CUT&Tag assays for protein-DNA interactions if studying C12orf60's potential role in transcriptional regulation

  • Proximity ligation assays to validate protein-protein interactions

• Genetic analysis approaches:

  • Examine potential associations between C12orf60 genetic variants and phenotypes using datasets from population studies

How can I troubleshoot weak or non-specific C12orf60 antibody signals?

When encountering issues with C12orf60 antibody performance, consider these troubleshooting strategies:

For immunohistochemistry applications specifically, optimal results have been demonstrated using a 1:100 dilution for human kidney and pancreatic tissue samples . For immunofluorescence, A549 cells have been successfully stained using a 1:100 dilution combined with Alexa Fluor 488-conjugated secondary antibodies .

How should I design experiments to study C12orf60's potential role in cell cycle regulation?

Based on C12orf60's putative involvement in cell cycle regulation, consider the following experimental design strategies:

• Cell synchronization experiments:

  • Synchronize cells at different cell cycle phases using methods like double thymidine block or nocodazole treatment

  • Analyze C12orf60 expression and localization across cell cycle phases using the validated antibody dilutions for IF (1:50-1:200)

  • Correlate with known cell cycle markers (e.g., cyclin proteins, Ki-67)

• Co-localization studies:

  • Perform dual immunofluorescence staining with C12orf60 (1:100 dilution) and cell cycle regulators

  • Analyze subcellular localization changes during different cellular states

  • Quantify co-localization using Pearson's correlation coefficient or Manders' overlap coefficient

• Functional perturbation:

  • Overexpress or knockdown C12orf60 and analyze effects on:

    • Cell cycle distribution (flow cytometry)

    • Expression of cyclins and CDKs (Western blot)

    • BrdU incorporation (proliferation)

    • Cellular morphology and viability

• Protein interaction studies:

  • Immunoprecipitate C12orf60 using the polyclonal antibody

  • Identify binding partners through mass spectrometry

  • Validate interactions with known cell cycle proteins

Include appropriate controls at each step, particularly positive controls from tissues with demonstrated C12orf60 expression such as kidney and pancreatic tissues .

What are the considerations for using C12orf60 antibodies in cancer research models?

When applying C12orf60 antibodies in cancer research, consider these methodological approaches:

• Expression analysis across cancer types:

  • Compare C12orf60 protein levels in tumor vs. adjacent normal tissues using IHC (1:20-1:200 dilution)

  • Correlate expression with clinical parameters and patient outcomes

  • Create tissue microarrays for high-throughput screening

• Cell line panel screening:

  • Screen diverse cancer cell lines for C12orf60 expression using Western blot (1:500-1:2000)

  • Correlate with cellular phenotypes (proliferation rate, invasiveness, drug resistance)

  • A549 lung carcinoma cells have shown detectable C12orf60 expression and can serve as a positive control

• Functional studies in cancer models:

  • Manipulate C12orf60 expression in cancer cell lines

  • Assess effects on:

    • Proliferation and apoptosis markers

    • Migration and invasion capabilities

    • Response to chemotherapeutic agents

    • In vivo tumor growth in xenograft models

• Potential biomarker investigation:

  • Evaluate C12orf60 expression in liquid biopsies using ELISA (1:2000-1:10000)

  • Assess correlation with disease progression and treatment response

  • Compare with established biomarkers

When interpreting cancer-related data, consider that C12orf60's relatively uncharacterized nature means hypotheses about its role should be validated through multiple experimental approaches rather than relying solely on antibody-based detection.

How can I integrate C12orf60 antibody data with genetic analysis approaches?

To develop a more comprehensive understanding of C12orf60 function, consider integrating antibody-based protein studies with genetic approaches:

• Genotype-phenotype correlations:

  • Analyze C12orf60 protein expression in samples with known genetic variants

  • Test whether genetic polymorphisms affect protein expression levels or cellular localization

  • Investigate potential associations between C12orf60 variants and disease susceptibility

• Multi-omics integration:

  • Correlate protein expression data (from antibody-based detection) with:

    • Transcriptomic data (RNA-seq)

    • Genomic data (genotyping, exome sequencing)

    • Epigenomic profiles (methylation patterns, histone modifications)

• CRISPR/Cas9 genome editing:

  • Generate C12orf60 knockout or knock-in cell lines

  • Validate antibody specificity using knockout controls

  • Characterize phenotypic changes resulting from gene modification

• Functional genomics approaches:

  • Consider CUT&Tag assays if investigating potential DNA-binding properties or chromatin associations

  • Combine with ChIP-seq if examining interactions with transcription factors

  • Validate findings using the C12orf60 antibody in orthogonal assays

These integrated approaches provide more robust evidence of C12orf60 function than any single method alone, which is particularly important given the protein's uncharacterized status.

What considerations exist for using C12orf60 antibodies in co-immunoprecipitation experiments?

When designing co-immunoprecipitation (Co-IP) experiments to identify C12orf60 interaction partners:

• Antibody selection:

  • Use the polyclonal C12orf60 antibody that has been Protein G purified to >95% purity

  • Consider whether the antibody was raised against the full protein (1-245AA) or a fragment

• Experimental conditions:

  • Optimize lysis buffer composition (NP-40, RIPA, or gentler formulations)

  • Include appropriate protease and phosphatase inhibitors

  • Consider crosslinking approaches for transient interactions

  • Use appropriate antibody amounts (typically 2-5 μg per mg of protein lysate)

• Controls:

  • Input control (5-10% of pre-immunoprecipitation lysate)

  • IgG isotype control immunoprecipitation

  • Reciprocal Co-IP with antibodies against suspected interaction partners

  • If available, C12orf60-depleted or knockout cells as negative controls

• Detection methods:

  • Western blot analysis using antibodies against predicted interaction partners

  • Mass spectrometry for unbiased identification of co-precipitated proteins

  • Validation of identified interactions using proximity ligation assays or FRET

• Result interpretation:

  • Consider that the polyclonal nature of the antibody may affect specificity

  • Validate key interactions through multiple methods

  • Assess biological relevance of identified interactions

This systematic approach helps identify genuine protein interactions while minimizing false positives that can arise in Co-IP experiments.

Can C12orf60 antibodies be adapted for use in CUT&Tag assays?

CUT&Tag (Cleavage Under Targets and Tagmentation) is an emerging technique for studying protein-DNA interactions with high sensitivity, particularly valuable for samples with limited cell numbers . Adapting C12orf60 antibodies for CUT&Tag requires several considerations:

• Antibody suitability assessment:

  • C12orf60 antibodies would need to maintain specificity under native/unfixed cell conditions

  • The polyclonal nature of available antibodies (like PACO36414) may provide better epitope recognition in the native state

  • Test antibody functionality in native chromatin conditions before proceeding

• Protocol adaptation:

  • Follow the general CUT&Tag workflow outlined in the search results :

    • Cell permeabilization without fixation

    • Primary antibody incubation (C12orf60 antibody)

    • Secondary antibody binding

    • pA-Tn5 transposome binding and tagmentation

  • Optimize antibody concentration (starting with 1:100 dilution as used in IF applications)

• Controls and validation:

  • Include known DNA-binding proteins as positive controls

  • Use IgG controls to establish background levels

  • Validate findings with orthogonal methods (ChIP-seq if sufficient material is available)

• Data analysis considerations:

  • Since C12orf60's DNA binding properties (if any) are uncharacterized, analyze data without prior assumptions

  • Look for enrichment patterns that might inform function

  • Compare with publicly available datasets of known transcription factors or chromatin modifiers

While CUT&Tag offers advantages for limited samples and provides high signal-to-noise ratios , the application to C12orf60 would be exploratory given its uncharacterized nature and would require thorough validation.

How can I quantitatively assess C12orf60 expression across different tissues or experimental conditions?

For rigorous quantitative analysis of C12orf60 expression:

• Western blot quantification:

  • Use validated dilutions (1:500-1:2000) of the C12orf60 antibody

  • Include calibration standards of known C12orf60 concentrations

  • Normalize to loading controls (β-actin, GAPDH)

  • Use digital image analysis software for densitometry

  • Ensure linear detection range by testing multiple exposure times

• Quantitative immunohistochemistry:

  • Use consistent staining protocols at validated antibody dilutions (1:20-1:200)

  • Employ digital pathology systems for quantification

  • Develop scoring systems (H-score, Allred score) for consistent evaluation

  • Include control tissues (kidney and pancreatic tissues) on each slide

• ELISA-based quantification:

  • Develop a sandwich ELISA using the C12orf60 antibody

  • Generate standard curves using recombinant C12orf60 protein

  • Follow manufacturers' recommended dilutions (1:2000-1:10000)

  • Validate assay precision with spike-recovery experiments

• Multiplexed protein analysis:

  • Consider techniques like Luminex or protein microarrays

  • Validate antibody performance in multiplex format

  • Include appropriate normalization controls

When analyzing data, consider using statistical approaches appropriate for the data distribution and experimental design. For non-normal distributions, consider rank-based inverse normal transformation, similar to approaches used in antibody analyses in population studies .

What are the current limitations in C12orf60 antibody research?

Current C12orf60 antibody research faces several key limitations:

• Limited functional characterization:

  • C12orf60 remains largely uncharacterized , making it difficult to interpret antibody-based findings in a functional context

  • The specific cellular roles and pathways involving C12orf60 require further elucidation

• Antibody validation challenges:

  • Limited availability of knockout models for definitive specificity validation

  • Potential cross-reactivity with related proteins

  • Variability between different antibody preparations and lots

• Application restrictions:

  • Validated applications are currently limited to ELISA, WB, IHC, and IF

  • Flow cytometry and other specialized applications may require additional validation

  • Species reactivity is primarily focused on human samples, with predicted but not fully validated reactivity in other species

• Data interpretation complexities:

  • Distinguishing specific from non-specific signals requires robust controls

  • Correlating protein expression with functional outcomes remains challenging

Addressing these limitations requires rigorous experimental design, comprehensive controls, and integration of multiple complementary approaches to strengthen confidence in research findings.

How might C12orf60 antibodies contribute to understanding disease mechanisms?

Despite current limitations, C12orf60 antibodies offer valuable opportunities for disease research:

• Cancer biology:

  • Given C12orf60's potential role in cell proliferation and apoptosis , antibody-based studies could reveal its expression patterns across cancer types

  • Correlation with clinical outcomes might identify prognostic biomarker potential

  • Mechanistic studies could elucidate its role in oncogenic or tumor-suppressive pathways

• Cell cycle disorders:

  • Antibody-based localization studies during cell cycle progression

  • Investigation of expression in disorders characterized by dysregulated cell division

  • Potential therapeutic target identification if functionally relevant

• Biomarker development:

  • ELISA-based detection in patient samples

  • Tissue microarray screening across disease cohorts

  • Correlation with genetic variants identified in population studies

• Drug discovery applications:

  • Target validation using antibody-based detection

  • Compound screening effects on C12orf60 expression or localization

  • Mechanism of action studies for drugs affecting cell proliferation pathways

As research progresses, C12orf60 antibodies will likely play an increasingly important role in uncovering this protein's functions in normal physiology and disease states, potentially leading to new diagnostic or therapeutic strategies.