ZSCAN9 Antibody

What is ZSCAN9 and what is its biological role?

ZSCAN9 (Zinc Finger and SCAN Domain-Containing Protein 9) is a transcription factor also known as ZNF193, PIG12, PRD51, and Cell proliferation-inducing gene 12 protein. The protein is believed to be involved in transcriptional regulation based on its structural domains . As a member of the zinc finger protein family, it likely binds to specific DNA sequences to influence gene expression patterns. The presence of the SCAN domain suggests potential protein-protein interactions with other transcription factors, possibly forming regulatory complexes that modulate cellular proliferation pathways .

What types of ZSCAN9 antibodies are currently available for research?

Several types of ZSCAN9 antibodies are available for research applications, including:

• Rabbit polyclonal antibodies that react with human and mouse ZSCAN9 (unconjugated format)

• Rabbit polyclonal antibodies with biotin conjugation

• Mouse polyclonal antibodies raised against full-length human ZNF193 protein (AA 1-394)

The choice between these depends on your experimental design, target species, and application requirements. Each antibody has been generated using different immunogens, which affects epitope recognition and potentially application performance.

What is the molecular identity of ZSCAN9 that antibodies target?

ZSCAN9 antibodies target the protein encoded by gene ID 7746, with UniProt accession number O15535 . The protein has several alias names including Zinc finger protein 193, PIG12, PRD51, and Cell proliferation-inducing gene 12 protein . The full protein sequence (for human) includes 394 amino acids with multiple zinc finger domains and a SCAN domain . When selecting antibodies, researchers should verify which region of the protein the antibody targets, as this impacts recognition of potential isoforms or post-translationally modified variants.

What applications are ZSCAN9 antibodies validated for?

ZSCAN9 antibodies have been validated for multiple applications with varying recommended protocols:

For optimal results, researchers should perform titration experiments to determine the ideal concentration for their specific experimental conditions, as recommended dilutions may vary based on sample types and detection methods .

How should I validate a ZSCAN9 antibody before use in my experiments?

A rigorous validation approach for ZSCAN9 antibodies should follow these steps:

• CRISPR/Cas9 knockout validation: Generate ZSCAN9 knockout cell lines using CRISPR/Cas9 in a cell type with high endogenous expression. Compare antibody reactivity between parental and knockout lines by immunoblotting to confirm specificity .

• Positive control identification: Use proteomics databases to identify cell lines with high ZSCAN9 expression for positive controls .

• Application-specific validation: Test the antibody in your specific application (WB, IHC, IF, etc.) with appropriate positive and negative controls .

• Cross-reactivity assessment: Evaluate potential cross-reactivity with related zinc finger proteins, particularly those with similar SCAN domains.

• Lot-to-lot consistency: When reordering the same antibody, perform abbreviated validation to ensure consistent performance between lots.

This systematic approach significantly increases confidence in antibody specificity and experimental reproducibility .

What are the optimal storage conditions for maintaining ZSCAN9 antibody activity?

For maximum stability and activity retention, ZSCAN9 antibodies should be stored according to these guidelines:

• Store at -20°C for long-term storage

• For rabbit polyclonal antibodies, aliquot and avoid repeated freeze/thaw cycles

• Some biotin-conjugated antibodies may benefit from -80°C storage for extended periods

• Store in buffer containing 50% glycerol to prevent freezing damage

• The rabbit polyclonal antibody is typically provided in 0.01M PBS, pH 7.4, with 0.03% Proclin-300 and 50% glycerol

Improper storage can lead to protein aggregation, degradation, and loss of epitope recognition, resulting in diminished performance in experimental applications.

How can I troubleshoot non-specific binding with ZSCAN9 antibodies?

When experiencing non-specific binding with ZSCAN9 antibodies, implement these methodological approaches:

• Increase blocking stringency: Extend blocking time or try alternative blocking agents (5% milk, 5% BSA, or commercial blocking buffers).

• Optimize antibody concentration: Perform a dilution series to identify the optimal antibody concentration that maximizes specific signal while minimizing background .

• Adjust washing conditions: Increase washing duration or add mild detergents (0.1% Tween-20) to reduce non-specific interactions.

• Pre-adsorb the antibody: Incubate diluted antibody with knockout or negative control lysates to remove cross-reactive antibodies.

• Validate specificity: Compare results with ZSCAN9 knockout controls to distinguish between specific and non-specific signals .

• Try alternative antibodies: Different antibodies targeting distinct epitopes may offer improved specificity in your particular application.

Rigorous validation using knockout controls remains the gold standard for confirming antibody specificity .

How can I use ZSCAN9 antibodies for co-immunoprecipitation studies?

For successful co-immunoprecipitation (co-IP) studies with ZSCAN9 antibodies:

• Antibody selection: Choose antibodies validated for immunoprecipitation applications. While specific IP validation for commercial ZSCAN9 antibodies is limited in the provided information, general antibody validation procedures can be applied .

• Cross-linking consideration: For transient or weak interactions, consider using membrane-permeable cross-linking agents prior to cell lysis.

• Buffer optimization: Use lysis buffers that preserve protein-protein interactions while efficiently extracting ZSCAN9 (typically containing 0.5-1% NP-40 or Triton X-100, 150mM NaCl, and protease inhibitors).

• Controls: Include:

  • IgG control from the same species as the ZSCAN9 antibody

  • ZSCAN9 knockout lysate as a negative control

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

• Validation: Confirm successful pull-down by immunoblotting a portion of the immunoprecipitate for ZSCAN9 before analyzing for interacting partners.

• Mass spectrometry analysis: For unbiased identification of interaction partners, analyze immunoprecipitates by mass spectrometry.

This approach allows for investigation of ZSCAN9's potential role in transcriptional complexes and regulatory networks.

How can I detect different ZSCAN9 isoforms using available antibodies?

To effectively distinguish between ZSCAN9 isoforms:

• Determine antibody epitope location: Review the immunogen information to identify which region of ZSCAN9 the antibody recognizes . The antibody in source targets the full-length protein (AA 1-394), potentially recognizing multiple isoforms.

• Isoform analysis: Compare experimental band patterns with expected molecular weights of known ZSCAN9 isoforms. The NCBI accession numbers (NP_001186408.1, NP_001186409.1) suggest multiple isoforms exist .

• High-resolution gel systems: Use gradient gels (4-12% or 4-20%) to improve separation of closely sized isoforms.

• Isoform-specific controls: Where possible, use recombinant isoforms or cells expressing individual isoforms as controls.

• Complementary techniques: Combine antibody detection with RT-PCR to correlate protein detection with isoform-specific mRNA expression.

• Knockout validation: Verify that all detected bands disappear in ZSCAN9 knockout samples to confirm they represent true isoforms rather than cross-reactive proteins .

This methodological approach helps distinguish between true isoforms and non-specific signals.

How can ZSCAN9 antibodies be used to investigate its role in transcriptional regulation?

ZSCAN9 is believed to function in transcriptional regulation , and these methodological approaches can help investigate this role:

• Chromatin immunoprecipitation (ChIP): Use validated ZSCAN9 antibodies to identify genomic binding sites, though antibodies must first be validated specifically for ChIP applications.

• Co-immunoprecipitation studies: Identify protein interaction partners within transcriptional complexes using protocols outlined in section 4.1.

• Immunofluorescence co-localization: Examine nuclear localization patterns and potential co-localization with other transcription factors or chromatin markers.

• Transcriptomic analysis with knockdown/knockout: Compare gene expression profiles between wild-type and ZSCAN9-depleted cells to identify regulated genes.

• Reporter assays: Use reporter constructs containing putative ZSCAN9 binding sites to assess direct transcriptional regulatory effects.

These approaches can help elucidate ZSCAN9's specific role in transcriptional networks and gene regulation pathways.

What considerations are important when using ZSCAN9 antibodies for immunohistochemistry?

For successful ZSCAN9 immunohistochemistry (IHC) applications:

• Antibody selection: Choose antibodies specifically validated for IHC. The rabbit polyclonal antibody has been tested for IHC with recommended dilutions of 1/20-1/200 .

• Antigen retrieval optimization: Test multiple antigen retrieval methods (heat-induced with citrate buffer, pH 6.0, or EDTA buffer, pH 9.0) to determine optimal epitope exposure conditions.

• Antibody validation: Use tissues from ZSCAN9 knockout models as negative controls to confirm staining specificity .

• Positive control selection: Based on expression data, identify tissues with known high ZSCAN9 expression to serve as positive controls.

• Signal amplification: For low-abundance expression, consider using signal amplification systems (e.g., tyramide signal amplification) while monitoring background levels.

• Counterstaining: Use nuclear counterstains like hematoxylin to assess subcellular localization, as ZSCAN9 is expected to show primarily nuclear localization.

Careful method optimization and rigorous controls ensure reliable and reproducible IHC results.