CDADC1 Antibody

What is CDADC1 and why is it important in research?

CDADC1 is an enzyme that catalyzes the deamination of cytidine and deoxycytidine into uridine and deoxyuridine, respectively. It plays a crucial role in maintaining balanced dNTP pools essential for DNA replication, repair, and other cellular processes. Recent studies suggest CDADC1 may have particular significance in testicular development and spermatogenesis . Unlike other cytidine deaminases, CDADC1 has a unique structure with two deaminase domains, making it an interesting target for researchers studying nucleotide metabolism and reproductive biology .

What applications are CDADC1 antibodies typically used for?

CDADC1 antibodies have been validated for multiple applications including:

• Western blotting for detecting native and recombinant CDADC1 in cell lysates

• Immunohistochemistry (IHC) on formalin-fixed paraffin-embedded tissues

• Immunofluorescence/Immunocytochemistry (IF/ICC)

• Enzyme-linked immunosorbent assay (ELISA)

• RNAi knockdown validation studies

Western blotting is particularly useful for detecting CDADC1's expected molecular weight of approximately 58 kDa, though the transfected form has been observed at 58.5 kDa .

What is the difference between monoclonal and polyclonal CDADC1 antibodies?

Monoclonal CDADC1 antibodies (like clone 1A2) recognize a single epitope, typically within a specific region of the protein (e.g., amino acids 423-514) . They offer high specificity but might be less robust to protein denaturation. Polyclonal CDADC1 antibodies recognize multiple epitopes across the protein, potentially providing stronger signals and greater tolerance to protein modifications or partial denaturation . For critical experiments, using both types can provide complementary data and validation.

What tissues show high CDADC1 expression for positive controls?

Testis tissue (both human and mouse) serves as an excellent positive control for CDADC1 antibody validation in immunohistochemistry applications . For cellular studies, several cell lines including RAW 264.7, NIH/3T3, and Saos-2 cells have demonstrated detectable CDADC1 expression and can be used as positive controls .

How can I optimize Western blot protocols for CDADC1 detection?

For optimal CDADC1 detection in Western blot experiments:

• Use fresh lysates from appropriate positive control cells (RAW 264.7, NIH/3T3, or 293T cells)

• Include GAPDH (36.1 kDa) as a loading and specificity control

• Be aware that recombinant CDADC1 with a GST tag will appear larger (~35.86 kDa for the partial recombinant plus 26 kDa for the GST tag)

• For validation studies, include both transfected and non-transfected lysates to confirm antibody specificity

When troubleshooting weak signals, consider that CDADC1 forms a homo-hexameric structure, which might affect epitope accessibility in some experimental conditions .

What are the critical parameters for CDADC1 immunohistochemistry?

For successful CDADC1 immunohistochemistry:

• Optimal antigen retrieval is critical – use TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0

• For monoclonal antibodies, a concentration of 1 μg/ml has been validated for human kidney tissue

• For polyclonal antibodies, dilutions between 1:50-1:500 are recommended

• Include human or mouse testis tissue as positive controls

• Perform parallel negative controls (secondary antibody only or isotype control)

These parameters ensure specific staining while minimizing background, which is particularly important when studying tissues with varying CDADC1 expression levels.

How can I validate CDADC1 antibody specificity in my experiments?

Comprehensive validation of CDADC1 antibody specificity should include:

• RNAi knockdown experiments – compare CDADC1 antibody staining in wild-type versus CDADC1-knockdown cells (using validated CDADC1 RNAi constructs like H00081602-R01V)

• Overexpression controls – test antibody on cells transfected with CDADC1 expression vectors

• Western blot analysis showing a single band at the expected molecular weight (~58 kDa)

• Testing reactivity against the recombinant immunogen

• Cross-species validation if working across different model organisms (human-mouse cross-reactivity has been confirmed)

This multi-pronged approach minimizes the risk of misinterpreting non-specific signals as genuine CDADC1 detection.

How can antibodies help investigate CDADC1's unique dual-domain structure?

CDADC1's distinctive structure featuring catalytically active C-terminal and regulatory N-terminal domains can be investigated using domain-specific antibodies:

• Select antibodies recognizing epitopes in specific domains (e.g., antibodies raised against amino acids 423-514 target the C-terminal domain)

• Use these antibodies in combination with site-directed mutagenesis (e.g., E400A mutation that abolishes catalytic activity)

• Employ co-immunoprecipitation followed by mass spectrometry to identify domain-specific binding partners

• Perform immunofluorescence to determine if domain accessibility changes under different cellular conditions

These approaches can provide insights into how CDADC1's unique structural arrangement contributes to its enzymatic function and regulation.

What methods can I use to study CDADC1's homo-hexameric quaternary structure?

The recently solved cryo-EM structures of CDADC1 at ~3-4.7Å resolution revealed its organization as a homo-hexamer arranged as a stack of two trimers. To investigate this quaternary structure:

• Use native PAGE and Western blotting with CDADC1 antibodies to detect the higher molecular weight complex

• Perform chemical crosslinking followed by SDS-PAGE and immunoblotting

• Use size exclusion chromatography combined with Western blot analysis

• Consider proximity ligation assays to visualize CDADC1 self-association in situ

• Compare wild-type to the E400A catalytically inactive mutant to determine if hexamer formation is activity-dependent

These methodologies can provide valuable insights into how CDADC1's quaternary structure relates to its function in nucleotide metabolism.

How can CDADC1 antibodies be used to investigate its role in nucleotide metabolism?

To study CDADC1's function in nucleotide metabolism:

• Combine CDADC1 immunoprecipitation with activity assays measuring the conversion of CTP/dCTP to UTP/dUTP

• Use immunofluorescence to co-localize CDADC1 with other nucleotide metabolism enzymes

• Perform ChIP-seq experiments to determine if CDADC1 associates with chromatin during DNA replication

• Use CDADC1 antibodies to track protein levels following treatment with nucleotide analogs or metabolism inhibitors

• Develop ELISA-based assays to quantify CDADC1 levels in response to changes in cellular nucleotide pools

These approaches can clarify CDADC1's unique activity on CTP and dCTP, which has not been previously reported in eukaryotes.

What experimental approaches can reveal CDADC1's role in reproductive biology?

Given CDADC1's potential importance in testicular development and spermatogenesis:

• Use immunohistochemistry with CDADC1 antibodies on testis sections at different developmental stages

• Perform co-localization studies with markers of spermatogonia, spermatocytes, and spermatids

• Analyze CDADC1 expression in fertile versus infertile tissue samples

• Investigate potential hormone-dependent regulation of CDADC1 expression

• Combine with genetic models (knockdown/knockout) to correlate CDADC1 levels with reproductive phenotypes

The validated reactivity of CDADC1 antibodies in both human and mouse testis tissues makes these approaches particularly feasible for reproductive biology research.

How do I address non-specific binding when using CDADC1 antibodies?

When encountering non-specific binding:

• Optimize blocking conditions – try 5% BSA instead of milk for Western blots

• Increase the stringency of wash steps by adding additional detergent (0.1-0.3% Tween-20)

• For IHC/ICC, include a protein blocking step with normal serum matching the secondary antibody host

• Titrate the primary antibody concentration (try 1:200-1:800 for ICC applications)

• Include appropriate controls (isotype controls for monoclonal antibodies)

• Consider pre-adsorption with the immunizing peptide if available

Careful optimization of these parameters can significantly improve signal-to-noise ratio in CDADC1 detection.

What are the best storage conditions for maintaining CDADC1 antibody activity?

To preserve CDADC1 antibody function:

• Store at -20°C in appropriate buffer (e.g., PBS with 0.02% sodium azide and 50% glycerol pH 7.3)

• Avoid repeated freeze-thaw cycles by preparing working aliquots

• For monoclonal antibodies, monitor for precipitates that might indicate denaturation

• Verify antibody performance periodically using positive control samples

• Follow manufacturer's recommendations for short-term storage during experiments

Proper storage ensures consistent antibody performance across experiments and extends shelf life.