C17orf64 Antibody

What is C17orf64 and what is its biological significance?

C17orf64 is an uncharacterized protein encoded by a gene mapping to human chromosome 17. While its specific function remains largely unknown, its location on chromosome 17 is notable as this chromosome contains crucial tumor suppressor genes including p53 and BRCA1 . Chromosome 17 makes up over 2.5% of the human genome with approximately 81 million bases encoding over 1,200 genes . Research interest in C17orf64 stems partly from its genomic neighborhood, which is associated with multiple diseases including various cancers, neurofibromatosis, Alexander disease, Birt-Hogg-Dube syndrome, and Canavan disease . Recent methylomic studies have identified C17orf64 as one of the top-performing loci in ovarian cancer analyses, suggesting potential roles in cancer development or progression .

What types of C17orf64 antibodies are available for research?

Multiple types of validated C17orf64 antibodies are available for research applications:

Most commercially available C17orf64 antibodies are produced in rabbit as polyclonal antibodies, which provide good sensitivity for detecting this relatively uncharacterized protein across multiple applications . Researchers should note that antibody validation levels vary between suppliers, with some products like the Prestige Antibodies® line undergoing enhanced validation through recombinant expression and orthogonal RNAseq verification .

What is the typical immunogen strategy for raising C17orf64 antibodies?

C17orf64 antibodies are typically generated using synthetic peptide fragments derived from the human C17orf64 sequence. For example, some antibodies utilize KLH conjugated synthetic peptides derived from the human C17orf64 sequence , while others target specific epitopes such as the sequence "LHSNISGMKERLSNMQTPGQGSPLPGQPRSQDHVKKDSLRELSQKPKLKRKRIKEAPETPETE" . The choice of immunogen significantly impacts the specificity and applications of the resulting antibody, with carefully selected regions producing antibodies with lower cross-reactivity to other proteins .

What validation methods ensure specificity of C17orf64 antibodies?

High-quality C17orf64 antibodies undergo multiple validation steps:

• Protein Array Screening: Testing against large panels (e.g., 364 human recombinant protein fragments) to confirm specificity

• Tissue Microarray Analysis: Validation through IHC using arrays of multiple normal human tissues (typically 44) and cancer tissues (approximately 20 common types)

• Enhanced Validation: Advanced verification through:

  • Recombinant expression systems

  • Orthogonal RNAseq correlation analysis

  • Testing in multiple species to confirm cross-reactivity claims

When selecting a C17orf64 antibody, researchers should review available validation data and prioritize antibodies validated through multiple methodologies. The Human Protein Atlas project provides extensive validation data for some C17orf64 antibodies, allowing researchers to examine actual immunostaining patterns across tissues and subcellular compartments .

How do I select the appropriate C17orf64 antibody for specific experimental applications?

Selection criteria should include:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF, etc.) with documented dilution ranges

• Species reactivity: Confirm cross-reactivity with your model organism (human, mouse, rat)

• Epitope location: Consider whether the recognized epitope might be masked in your experimental system

• Validation depth: Prioritize antibodies with comprehensive validation data, especially those tested across numerous tissues

• Buffer compatibility: Ensure the antibody formulation is compatible with your experimental buffers and fixation methods

For specific applications, recommended antibody dilutions typically range from 1:300-5000 for Western blotting, 1:200-400 for immunohistochemistry on paraffin sections, and 1:50-200 for immunofluorescence on paraffin sections .

What are the optimal sample preparation protocols for C17orf64 antibody applications?

For Immunohistochemistry (Paraffin Sections):

• Fix samples with paraformaldehyde

• Embed in paraffin

• Perform antigen retrieval by boiling in sodium citrate buffer (pH 6.0) for 15 minutes

• Block endogenous peroxidase activity with 3% hydrogen peroxide for 30 minutes

• Apply blocking buffer (normal goat serum) at 37°C for 20 minutes

• Incubate with primary antibody overnight at 4°C (typical dilution 1:200-1:1000)

• Apply conjugated secondary antibody followed by DAB staining

For Western Blotting:

• Use standard protein extraction methods appropriate for your tissue/cell type

• Determine optimal antibody concentration (typically 0.04-0.4 μg/mL or dilutions of 1:300-1:5000)

• Include appropriate blocking reagents to minimize background

• Optimize incubation time and temperature based on signal strength and specificity

What are common troubleshooting strategies for C17orf64 antibody experiments?

When optimizing protocols, remember that storage conditions significantly impact antibody performance. Most C17orf64 antibodies should be stored at -20°C, and repeated freeze/thaw cycles should be avoided .

How are C17orf64 antibodies utilized in cancer biomarker research?

C17orf64 has emerged as a significant locus in methylomic analyses of ovarian cancers . Researchers combine C17orf64 antibodies with methylation-specific techniques to:

• Correlate protein expression with methylation status: By comparing immunohistochemical staining patterns with methylation results obtained through bisulfite sequencing or methylation arrays

• Identify potential biomarkers: C17orf64 was identified among eight top-performing loci (along with c6orf174, IRX2, TUBB6, PTPRN, OTX2, LOC200726, and NEUROD1) in ovarian cancer studies

• Develop diagnostic assays: Researchers have developed SMART-MSP (Sensitive Melting Analysis after Real Time-Methylation Specific PCR) assays for C17orf64 methylation with clinical diagnostic potential

The integration of protein-level detection via antibodies with epigenetic analyses provides a comprehensive understanding of C17orf64's potential role in cancer development and progression.

What methylation-based techniques have been paired with C17orf64 antibody studies?

For comprehensive methylomic analysis involving C17orf64:

• Bisulfite Treatment: DNA samples undergo bisulfite conversion before analysis

• Illumina's Infinium Human MethylationEPIC BeadChip Analysis: Processed through functional normalization algorithms implemented in packages like minfi from Bioconductor

• SMART-MSP Assays: Performed using:

  • Bisulfite-treated DNA

  • Evagreen dye

  • Specific primers (300 nM forward and reverse)

  • Fluorescein reference dye

  • Platinum Taq® DNA Polymerase

  • Thermocycling conditions: 5 minutes at 95°C, followed by 50 cycles of 95°C for 30 seconds, assay-specific annealing temperature for 30 seconds, and 72°C for 30 seconds

How do C17orf64 expression patterns correlate with chromosome 17-associated diseases?

While C17orf64's specific role remains to be fully characterized, its location on chromosome 17 places it in proximity to genes associated with multiple diseases:

• Cancer connections: Chromosome 17 contains tumor suppressors p53 and BRCA1, both directly involved in DNA repair. Malfunction or loss of p53 expression is associated with malignant cell growth and Li-Fraumeni syndrome, while BRCA1 is linked to early-onset breast cancer and predisposition to cancers of the ovary, colon, prostate, and fallopian tubes

• Neurological disorders: Chromosome 17 is linked to neurofibromatosis (characterized by neural and epidermal lesions with dysregulated Schwann cell growth), Alexander disease, and Canavan disease

• Other genetic conditions: Associations with Birt-Hogg-Dube syndrome have been reported

Research utilizing C17orf64 antibodies in these disease contexts helps establish whether this uncharacterized protein has functional relationships with these better-characterized disease pathways.

What are the recommended quantification approaches for C17orf64 immunoassays?

For SMART-MSP (methylation) analysis:

• Analyze results using software like CFX Manager v3.1

• Use regression to obtain the mean Cq for each sample

• Calculate Percent Methylated Reference (PMR) values using the formula:
  PMR = [# methylated copies marker] / [# copies β-Actin = 1000] × 100%

• Generate receiver-operating characteristic (ROC) analysis and area under the curve (AUC) values

• Perform statistical analyses using R statistical software suite with linear regression models

For Immunohistochemistry quantification:

• Score staining intensity (typically on a 0-3 scale)

• Assess percentage of positive cells

• Calculate H-score or other composite metrics depending on research question

• Compare across tissues or conditions using appropriate statistical tests

How should researchers interpret C17orf64 expression in the context of methylation studies?

When interpreting combined antibody and methylation data for C17orf64:

• Establish baseline expression: Use normal tissue controls to establish baseline expression patterns across different cell types and tissues

• Correlate with methylation status: Determine whether protein expression inversely correlates with promoter methylation (the typical pattern for gene silencing)

• Consider context-specific factors: Evaluate whether expression patterns differ between tissue types or disease states

• Validate findings with multiple methods: Confirm key results using orthogonal techniques (e.g., immunohistochemistry, Western blotting, qRT-PCR)

• Interpret based on minimum detection thresholds: Consider assay sensitivity limitations, such as the established minimum specificity/sensitivity for assay development (e.g., EAF of 0.2% or 1 in 500 as determined using linear regression of the standard curve)

The integration of protein-level detection with methylation analysis provides insights into the regulatory mechanisms potentially affecting C17orf64 expression in normal and disease states.