znf830 Antibody

What is ZNF830 and why is it important in research?

ZNF830, also known as CCDC16 (coiled-coil domain containing 16) or OMCG1 (orphan maintenance of genome 1), is a zinc finger protein encoded by a gene located on chromosome 17 . This protein plays significant roles in cellular processes including DNA repair pathways, splicing mechanisms, and genome maintenance. Understanding ZNF830 function is particularly relevant for cancer research, developmental biology, and studies of genomic integrity. The protein contains coiled-coil domains that facilitate protein-protein interactions, making antibody-based detection methods vital for exploring its interactome and cellular localization .

What are the key specifications of commercially available ZNF830 antibodies?

ZNF830 antibodies are primarily available as rabbit polyclonal antibodies, with several validated options from different manufacturers. Most commonly used antibodies target the full ZNF830 protein or specific epitopes. Key specifications include:

These antibodies typically recognize the human protein with high specificity, while also showing cross-reactivity with rat samples, making them versatile for comparative studies .

How do I determine the optimal working dilution for ZNF830 antibody in my experiment?

Determining the optimal working dilution requires systematic titration rather than relying solely on manufacturer recommendations. While typical Western blot dilutions range from 1:1000 to 1:6000 , the optimal concentration depends on:

• Sample type (cell line vs. tissue)

• Expression level of ZNF830 in your specific samples

• Detection method (chemiluminescence, fluorescence)

• Incubation conditions (time and temperature)

To optimize:

• Begin with manufacturer's recommended range (e.g., 1:1000-1:6000)

• Perform a dilution series experiment using positive control samples (HeLa cells or rat testis tissue are validated positive controls)

• Include appropriate negative controls

• Assess signal-to-noise ratio at each dilution

• Select the dilution that provides clear specific bands with minimal background

User validation data indicates successful results at 1:5000 dilution with 1-hour room temperature incubation for Western blot applications , but this should be verified for your specific experimental setup.

What are the validated applications for ZNF830 antibodies and their specific protocols?

ZNF830 antibodies have been validated for several applications, each requiring specific optimization:

Western Blot (WB):

• Primary antibody dilution: 1:1000-1:6000

• Incubation: 1-1.5 hours at room temperature

• Validated positive controls: HeLa cells, rat testis tissue

• Expected band: 42 kDa (primary band), with possible additional weak bands

• Standard protocol involves SDS-PAGE followed by transfer to membrane, blocking, overnight primary antibody incubation, and detection

Immunohistochemistry (IHC) and Immunocytochemistry (ICC):

• Multiple antibodies have been validated for these applications, though specific protocols may vary by manufacturer

• Fixation method and antigen retrieval steps significantly impact results

• Atlas Antibodies products have validated IHC and ICC-IF protocols

ELISA:

• Several antibodies including Proteintech's 25618-1-AP have been validated for ELISA applications

• Specific protocols are typically available from manufacturers

When designing experiments, it's advisable to reference specific protocol documents available from antibody suppliers for detailed methodology, as slight variations in procedure can significantly impact results .

How should I prepare and store ZNF830 antibody samples to maintain reactivity?

Proper handling and storage are critical for maintaining antibody performance over time:

These practices will help ensure consistent performance and reproducibility in experiments using ZNF830 antibodies.

What controls should be included when using ZNF830 antibodies?

A robust experimental design with appropriate controls is essential for reliable interpretation of ZNF830 antibody results:

Positive controls:

• HeLa cell lysates have been validated as reliable positive controls for ZNF830 detection

• Rat testis tissue has also been verified as a positive control

• Include samples with known ZNF830 expression levels to validate antibody performance

Negative controls:

• Primary antibody omission control (all reagents except primary antibody)

• Isotype control (irrelevant rabbit IgG at the same concentration)

• ZNF830 knockout or knockdown samples (if available)

• Pre-absorption control (antibody pre-incubated with immunizing peptide)

Loading controls:

• Include appropriate housekeeping protein controls (β-actin, GAPDH, etc.)

• For subcellular localization studies, include markers for relevant compartments

Cross-reactivity assessment:

• When working with non-human samples, note the sequence homology: mouse (88%), rat (87%)

• Validate new species applications not previously tested by manufacturers

Properly designed controls help distinguish specific signals from non-specific background and validate antibody specificity in your experimental system.

Why might I observe multiple bands or bands at unexpected molecular weights when using ZNF830 antibody?

Multiple bands or unexpected molecular weights are common challenges when working with antibodies. For ZNF830 specifically:

To differentiate specific from non-specific bands, consider running parallel samples with ZNF830 knockdown/knockout and wild-type controls to identify which bands disappear with target depletion.

How can I optimize signal-to-noise ratio when using ZNF830 antibodies?

Optimizing signal-to-noise ratio is crucial for generating clean, interpretable data:

• Blocking optimization:

  • Test different blocking agents (5% BSA, 5% non-fat milk, commercial blockers)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Include 0.1-0.3% Tween-20 in blocking buffer to reduce non-specific binding

• Antibody dilution adjustment:

  • Titrate primary antibody concentration (1:1000-1:6000 for Western blot)

  • One validated user reported excellent results at 1:5000 dilution

  • Optimize secondary antibody concentration independently

• Washing stringency:

  • Increase number of washes (5-6 washes of 5-10 minutes each)

  • Use higher detergent concentration in wash buffer (0.1-0.3% Tween-20)

  • Consider more stringent wash buffers for high background (add 500 mM NaCl)

• Incubation conditions:

  • Adjust primary antibody incubation (overnight at 4°C vs. 1-1.5 hours at room temperature)

  • Perform all incubations with gentle agitation to ensure even distribution

• Sample quality:

  • Use fresh samples and avoid repeated freeze-thaw cycles

  • Include protease and phosphatase inhibitors in lysis buffers

  • Ensure complete protein denaturation for Western blot applications

When working with ZNF830 antibodies specifically, note that a 1:5000 dilution with 1 hour incubation at room temperature has been positively validated by users for Western blot applications .

What are common challenges when using ZNF830 antibodies across different species?

Cross-species reactivity is an important consideration when planning experiments:

• Validated species reactivity:

  • Human: Confirmed by multiple manufacturers

  • Rat: Confirmed reactivity

  • Mouse: Some antibodies show predicted reactivity based on sequence homology

• Sequence conservation considerations:

  • The antigen sequence identity is approximately 88% for mouse and 87% for rat orthologs

  • This high degree of conservation suggests potential cross-reactivity, but validation is necessary

• Challenges with non-validated species:

  • Higher background or non-specific binding

  • Altered epitope recognition due to species-specific post-translational modifications

  • Different expression levels across species affecting detection sensitivity

• Optimization strategies for cross-species applications:

  • Increase antibody concentration for species with lower sequence homology

  • Optimize blocking and washing conditions more stringently

  • Consider using species-specific positive control samples

  • Validate with knockdown/knockout controls in the species of interest

• Application-specific considerations:

  • Western blot typically requires less epitope conservation than immunohistochemistry

  • For IHC/ICC applications in non-validated species, additional validation steps are strongly recommended

When working across species, begin with the established protocols for validated species and systematically adjust conditions to optimize for your specific experimental system.

How can I use ZNF830 antibodies to investigate protein-protein interactions and complexes?

ZNF830 antibodies can be powerful tools for studying protein-protein interactions through various methodologies:

• Co-immunoprecipitation (Co-IP):

  • ZNF830 antibodies can be used to pull down native protein complexes

  • Recommended starting dilution: 2-5 μg antibody per 500 μg total protein

  • Pre-clear lysates to reduce non-specific binding

  • Validate using reciprocal IP with antibodies against suspected interaction partners

  • Consider cross-linking IP for transient or weak interactions

• Proximity Ligation Assay (PLA):

  • Useful for detecting protein interactions in situ

  • Requires ZNF830 antibody raised in a different host species than the interaction partner antibody

  • Offers higher sensitivity than conventional co-localization studies

• Chromatin Immunoprecipitation (ChIP):

  • As ZNF830 is a nuclear protein, ChIP can identify DNA binding sites or chromatin associations

  • Requires optimization of cross-linking conditions and sonication parameters

  • Recommended starting point: 2-5 μg antibody per ChIP reaction

• Immunofluorescence co-localization:

  • Use ZNF830 antibodies in combination with markers for specific subcellular compartments

  • particularly nuclear compartments or DNA damage repair foci

  • Requires high-quality antibodies validated for ICC-IF applications

• Mass spectrometry approaches:

  • Immunoprecipitate ZNF830 complexes for mass spectrometry analysis

  • Consider RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) for protein complex identification

When designing these experiments, note that ZNF830 has been identified in complexes involved in DNA damage repair and RNA processing, suggesting potential interaction partners in these pathways.

What are the considerations for using ZNF830 antibodies in studies of DNA damage and repair pathways?

ZNF830 has been implicated in DNA damage response pathways, making antibodies against this protein valuable for studying genomic integrity:

• Experimental design considerations:

  • Include appropriate DNA damage induction controls (IR, UV, chemical agents)

  • Time-course experiments can capture dynamic recruitment to damage sites

  • Co-staining with established DNA damage markers (γH2AX, 53BP1, RAD51)

  • Cell cycle synchronization may be necessary to control for cell cycle-dependent effects

• Immunofluorescence applications:

  • ZNF830 antibodies can be used to track protein recruitment to DNA damage sites

  • Typical dilution for IF applications: start with manufacturer recommendations and optimize

  • Resolution considerations: consider super-resolution microscopy for precise localization

• Chromatin fractionation:

  • Biochemical fractionation followed by Western blot can detect ZNF830 recruitment to chromatin

  • Expected enrichment in chromatin fraction following DNA damage induction

  • Include positive controls (known chromatin-bound proteins) and loading controls

• Proximity-based labeling:

  • BioID or APEX2 fusion proteins can identify proteins proximal to ZNF830 at damage sites

  • Validation with co-IP using ZNF830 antibodies is recommended

• ChIP-seq applications:

  • Optimize ChIP conditions specifically for ZNF830

  • Include appropriate controls for antibody specificity

  • Consider ChIP-seq before and after DNA damage induction

When interpreting results, it's important to consider the potential dual roles of ZNF830 in both DNA repair and RNA processing pathways, which may complicate the analysis of localization patterns.

How can I validate ZNF830 antibody specificity for advanced applications?

For advanced applications, thorough validation of antibody specificity is essential:

• Genetic validation approaches:

  • CRISPR/Cas9 knockout: Generate ZNF830 knockout cells and confirm loss of signal

  • siRNA/shRNA knockdown: Partial reduction in protein should correlate with reduced signal intensity

  • Overexpression: Transfection with tagged ZNF830 should show co-localization with antibody staining

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of ZNF830

  • Consistent results with multiple antibodies increase confidence in specificity

  • Compare polyclonal (broader epitope recognition) with monoclonal (single epitope) antibodies

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide

  • Specific signals should be blocked while non-specific signals remain

  • Requires access to the immunizing peptide or recombinant protein

• Mass spectrometry validation:

  • Immunoprecipitate with ZNF830 antibody and analyze by mass spectrometry

  • Confirm presence of ZNF830 and characterize any co-precipitating proteins

  • Particularly valuable for validating antibodies used in interaction studies

• Orthogonal detection methods:

  • Compare results with tagged recombinant ZNF830 detected via the tag

  • Correlation between tag signal and antibody signal validates specificity

  • mRNA expression data should generally correlate with protein detection levels

• Application-specific validations:

  • For ChIP applications: perform ChIP-qPCR on positive and negative control regions

  • For tissue staining: compare with in situ hybridization patterns

  • For proximity labeling: confirm BioID or APEX2 results with co-IP using antibodies

These validation strategies should be tailored to the specific research question and application to ensure robust and reproducible results.