CDC73 Antibody, Biotin conjugated

What is CDC73 protein and why is it important in research?

CDC73, also known as parafibromin or HRPT2, is a 60 kDa protein that functions as a component of the PAF complex (PAFc), which controls RNA polymerase II-mediated general transcription . This protein has gained significant research interest due to its implications in parathyroid carcinoma and familial autosomal dominant hyperparathyroidism-jaw tumour syndrome (HPT-JT) . CDC73 plays a critical role in regulating mRNA stability, particularly of p53, thereby influencing p53-mediated apoptosis . Methodologically, studying CDC73 requires specific antibodies that can accurately detect and isolate this protein in various experimental contexts.

What are the main applications for biotin-conjugated CDC73 antibodies?

Biotin-conjugated CDC73 antibodies serve multiple research purposes with distinct methodological advantages:

When designing experiments, researchers should conduct titration experiments to determine optimal antibody concentrations for their specific samples and detection systems.

How do I distinguish between CDC73 isoforms using biotin-conjugated antibodies?

To distinguish between CDC73 isoforms, consider the following methodological approach:

• Verify the epitope recognized by your biotin-conjugated antibody by consulting the manufacturer's data sheet. Some antibodies target regions corresponding to the full-length Human NT5E or specific sequence segments (e.g., Q119-R520 as noted for some recombinant immunogens) .

• Perform Western blot analysis using positive controls with known expression of different isoforms. The expected molecular weight of the primary CDC73 protein is approximately 60 kDa .

• Consider using higher resolution SDS-PAGE (10-12%) to separate closely sized isoforms.

• For validation, combine your biotin-conjugated antibody with other antibodies recognizing different epitopes of CDC73 in parallel experiments.

How might the biotin conjugation affect CDC73 antibody binding to its cytoplasmic versus nuclear targets?

The biotin conjugation of CDC73 antibodies presents complex considerations for targeting differentially localized CDC73 proteins:

CDC73 functions in both nuclear and cytoplasmic compartments, with research suggesting distinct roles in each location. In the nucleus, CDC73 participates in the PAF complex for transcriptional regulation, while in the cytoplasm, it interacts with eEF1Bγ and hSki8 to regulate p53 mRNA stability . When using biotin-conjugated antibodies:

• Nuclear targeting may be affected by steric hindrance from the biotin molecule, particularly if the conjugation occurs near the epitope recognition site.

• For cytoplasmic CDC73 detection, researchers should verify antibody penetration by comparing permeabilization protocols (e.g., 0.1-0.5% Triton X-100 vs. 0.1% saponin).

• Quantitative analysis of nuclear vs. cytoplasmic signals should include appropriate controls to account for potential differences in antibody accessibility to different cellular compartments.

• When studying CDC73's interaction with p53 mRNA in the cytoplasm, consider using RNA-immunoprecipitation techniques with biotin-conjugated antibodies, followed by streptavidin pull-down for enhanced specificity .

What experimental controls are essential when using biotin-conjugated CDC73 antibodies to study cancer-related mutations?

When investigating cancer-related CDC73 mutations such as K34Q , implement these critical controls:

Additionally, when studying the K34Q mutation specifically, consider that this mutation enhances association with p53 mRNA , potentially altering epitope accessibility for certain antibodies.

How can I optimize immunoprecipitation protocols using biotin-conjugated CDC73 antibodies for protein-RNA interaction studies?

For studying CDC73's interactions with p53 mRNA or other RNA targets , optimize your immunoprecipitation protocol:

• Cross-linking optimization: Test formaldehyde concentrations (0.1-1%) and incubation times (5-15 minutes) to capture transient protein-RNA interactions without over-fixation.

• Antibody-to-bead ratio: For biotin-conjugated antibodies, pre-determine the optimal antibody-to-streptavidin bead ratio (typically starting with 1-5 μg antibody per 50 μl of bead slurry).

• Buffer composition: Include RNase inhibitors (40 U/μl) and adjust salt concentration (150-500 mM NaCl) to maintain specificity while preserving interactions.

• Elution strategy: For biotin-conjugated antibodies, consider competitive elution with biotin or direct RNA extraction from beads to avoid protein contamination.

• Sequential immunoprecipitation: To study CDC73's interaction with both eEF1Bγ and hSki8 , perform sequential IPs using differently labeled antibodies.

What are the optimal fixation and permeabilization protocols for using biotin-conjugated CDC73 antibodies in immunocytochemistry?

Fixation and permeabilization protocols significantly impact biotin-conjugated CDC73 antibody performance:

• Fixation comparison:

  • Paraformaldehyde (4%, 10-15 minutes): Preserves morphology but may mask some epitopes

  • Methanol (-20°C, 10 minutes): Better for nuclear antigen detection

  • Hybrid approach (2% PFA followed by methanol): Often optimal for both cytoplasmic and nuclear CDC73 detection

• Permeabilization optimization:

  • For biotin-conjugated antibodies targeting CDC73, use 0.2-0.5% Triton X-100 for balanced nuclear and cytoplasmic detection

  • Extend permeabilization time (15-30 minutes) when working with dense tissues

• Antigen retrieval:

  • For paraffin-embedded tissues, citrate buffer (pH 6.0) heat-mediated retrieval improves detection of CDC73 in both nuclear and cytoplasmic compartments

• Signal amplification:

  • Leverage biotin conjugation with streptavidin-HRP or streptavidin-fluorophore systems

  • Use tyramide signal amplification for ultra-sensitive detection of low-abundance CDC73

How should I troubleshoot inconsistent Western blot results when using biotin-conjugated CDC73 antibodies?

When facing inconsistent Western blot results with biotin-conjugated CDC73 antibodies, apply this systematic troubleshooting approach:

• Sample preparation issues:

  • CDC73 is susceptible to proteolytic degradation; use fresh protease inhibitor cocktails

  • Include phosphatase inhibitors as CDC73 function may be regulated by phosphorylation

  • Test different lysis buffers (RIPA vs. NP-40) as CDC73 extraction efficiency varies by buffer type

• Detection system optimization:

  • For biotin-conjugated antibodies, excessive endogenous biotin can cause background; block with avidin/biotin blocking kits

  • Use streptavidin-HRP at appropriate dilution (typically 1:1000-1:5000)

  • Validate detection with alternative systems (e.g., direct HRP-conjugated secondary antibodies)

• Membrane optimization:

  • PVDF membranes may provide better results than nitrocellulose for CDC73 detection

  • Optimize blocking conditions (5% BSA often superior to milk for phospho-specific detection)

• Molecular weight verification:

  • CDC73/parafibromin should appear at approximately 60 kDa

  • Verify specificity with positive control lysates

What is the most reliable approach for quantifying CDC73 expression levels using biotin-conjugated antibodies in flow cytometry?

For accurate quantification of CDC73 expression by flow cytometry:

• Standardization protocol:

  • Use calibration beads with known quantities of biotin-binding sites

  • Establish a standard curve relating fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF)

• Signal normalization:

  • Include consistent positive controls (cell lines with stable CDC73 expression)

  • Use isotype-matched biotin-conjugated controls at identical concentrations

  • Apply fluorescence minus one (FMO) controls to establish gating boundaries

• Multi-parameter analysis:

  • When studying CDC73's relationship to p53-mediated processes , incorporate co-staining for cell cycle markers and apoptosis indicators

  • Implement compensation matrices for spectral overlap when using multiple fluorochromes

• Data analysis:

  • Report median fluorescence intensity rather than mean values for non-normally distributed expression patterns

  • Use ratio of specific signal to isotype control rather than raw fluorescence values for cross-experimental comparisons

How can biotin-conjugated CDC73 antibodies be optimized for studying parathyroid carcinoma and HPT-JT syndrome?

CDC73 mutations are heavily implicated in parathyroid carcinoma and hyperparathyroidism-jaw tumour syndrome , requiring specialized methodological approaches:

• Tissue-specific protocol modifications:

  • For parathyroid tissues, extend antigen retrieval times (20-30 minutes)

  • Use lower antibody concentrations (0.25-0.5 μg/ml) for immunofluorescence to reduce background

  • Consider dual staining with parathyroid hormone (PTH) antibodies for co-localization studies

• Mutation-specific detection strategies:

  • Design comparative analyses between wild-type and CDC73 mutant tissues

  • Implement antibodies that specifically recognize wild-type epitopes (allowing detection of loss of expression)

  • For biotin-conjugated antibodies, optimize signal-to-noise ratio through titration experiments

• Clinical correlation techniques:

  • Establish scoring systems for CDC73 immunoreactivity (0-3+ scale)

  • Compare nuclear versus cytoplasmic staining patterns

  • Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma

What methodological approaches can integrate biotin-conjugated CDC73 antibodies with p53 pathway analysis?

Given CDC73's role in p53 mRNA stability and apoptosis regulation , consider these integrated approaches:

• Co-immunoprecipitation strategy:

  • Use biotin-conjugated CDC73 antibodies for initial pull-down

  • Analyze associated p53 mRNA through RT-qPCR

  • Quantify eEF1Bγ and hSki8 co-precipitation through Western blotting

• Sequential immunofluorescence protocol:

  • Implement multi-color immunofluorescence for CDC73, p53, and downstream apoptotic markers

  • Use biotin-conjugated CDC73 antibody with streptavidin-conjugated fluorophores compatible with other detection channels

  • Apply spectral unmixing for channels with overlapping emission spectra

• Functional validation approach:

  • Combine CDC73 detection with apoptosis assays (TUNEL, Annexin V)

  • Correlate CDC73 subcellular localization with p53 protein levels

  • Implement RNA-immunoprecipitation to directly measure CDC73-p53 mRNA interactions under different experimental conditions

How can biotin-conjugated CDC73 antibodies be incorporated into single-cell analysis platforms?

For integrating CDC73 antibodies into single-cell technologies:

• Mass cytometry (CyTOF) adaptation:

  • Conjugate CDC73 antibodies with isotope-labeled streptavidin for metal-based detection

  • Optimize antibody concentration for signal-to-noise ratio in multiplex panels

  • Include established markers for cell cycle and apoptosis to correlate with CDC73 expression

• Single-cell sequencing integration:

  • Implement CITE-seq protocols using biotin-conjugated CDC73 antibodies and oligonucleotide-tagged streptavidin

  • Correlate CDC73 protein levels with transcriptomic profiles

  • Apply computational methods to identify CDC73-associated gene expression networks

• Imaging mass cytometry:

  • Use biotin-conjugated CDC73 antibodies with metal-labeled streptavidin for spatial proteomics

  • Analyze subcellular localization patterns in relation to tissue architecture

  • Implement machine learning algorithms for pattern recognition across tissue sections

What are the optimal approaches for studying CDC73 post-translational modifications using biotin-conjugated antibodies?

To investigate CDC73 post-translational modifications:

• Phosphorylation analysis strategy:

  • Combine biotin-conjugated general CDC73 antibodies with phospho-specific antibodies

  • Implement phosphatase treatments as controls

  • Use Phos-tag gels to separate phosphorylated from non-phosphorylated CDC73 forms

• Ubiquitination detection method:

  • Perform sequential immunoprecipitation with biotin-conjugated CDC73 antibodies followed by ubiquitin detection

  • Include proteasome inhibitors (MG132) in experimental design

  • Compare wild-type CDC73 with K34Q mutant for differences in ubiquitination patterns

• Acetylation investigation approach:

  • Treat samples with histone deacetylase inhibitors to preserve acetylation

  • Use biotin-conjugated CDC73 antibodies for initial enrichment followed by acetyl-lysine antibody detection

  • Consider mass spectrometry to map specific acetylation sites