ZNF583 Antibody

What is ZNF583 and what are its key characteristics?

ZNF583 (Zinc finger protein 583) is a member of the krueppel C2H2-type zinc-finger protein family that is believed to be involved in transcriptional regulation . The protein has a molecular weight of approximately 66 kDa and is encoded by a gene located on chromosome 19q13.43 in humans . ZNF583 primarily localizes to the nucleus, which is consistent with its presumed function in transcriptional regulation .

The protein contains zinc finger domains that likely mediate DNA binding, allowing it to potentially regulate the expression of target genes. Recent research has suggested that ZNF583 expression may be associated with cancer progression and therapy response, making it an interesting target for oncology research .

What types of ZNF583 antibodies are commercially available for research?

Most commercially available ZNF583 antibodies are rabbit polyclonal antibodies generated against synthetic peptides or recombinant proteins corresponding to human ZNF583 . These antibodies are typically offered in unconjugated form, though some may be available with conjugates for specific applications. The antibodies are generally purified using peptide affinity chromatography methods to ensure specificity .

For optimal experimental design, researchers should note that these antibodies have been validated primarily for human samples, with some showing cross-reactivity to mouse and rat ZNF583 orthologs with varying degrees of sequence identity (approximately 62% for mouse and 55% for rat) .

What applications are ZNF583 antibodies validated for?

ZNF583 antibodies have been validated for several standard protein detection methods:

When designing experiments, researchers should perform their own validation and optimization steps, as performance can vary between antibody lots and experimental conditions. Using appropriate positive and negative controls is essential for reliable results.

What are the recommended storage and handling protocols for ZNF583 antibodies?

To maintain antibody activity and prevent degradation, the following storage and handling guidelines are recommended:

• For short-term storage (days), store at 4°C

• For long-term storage, keep at -20°C

• Avoid repeated freeze-thaw cycles to prevent antibody degradation

• ZNF583 antibodies are typically supplied in storage buffers containing:

  • PBS (phosphate-buffered saline)

  • Sodium azide (0.02-0.09%) as a preservative

  • Glycerol (typically 40%) or sucrose (2%) as cryoprotectants

• When working with lyophilized antibodies, reconstitute to 1 mg/mL concentration and aliquot to minimize freeze-thaw cycles

How should ZNF583 antibody specificity be validated in experimental settings?

Validating antibody specificity is crucial for obtaining reliable results. For ZNF583 antibodies, consider the following validation approaches:

• Blocking experiments: Use recombinant ZNF583 protein fragments (such as aa 63-138) as competitive inhibitors. Pre-incubate the antibody with a 100x molar excess of the protein fragment for 30 minutes at room temperature before application to your sample .

• Knockdown/knockout validation: Compare antibody reactivity in samples with normal ZNF583 expression versus samples where ZNF583 has been knocked down or knocked out using siRNA or CRISPR-Cas9.

• Multiple antibody validation: Use antibodies from different sources or those recognizing different epitopes of ZNF583 to confirm consistent results.

• Western blot analysis: Verify that the detected band corresponds to the expected molecular weight (66 kDa) .

• Positive and negative control tissues: Test the antibody on tissues known to express ZNF583 positively and negatively .

These validation methods should be performed in the context of your specific experimental conditions and sample types to ensure reliable results.

What are the optimal experimental conditions for Western blot detection of ZNF583?

For optimal Western blot detection of ZNF583, consider the following methodological recommendations:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • Denature samples thoroughly in reducing conditions (with β-mercaptoethanol or DTT)

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels (appropriate for 66 kDa proteins)

  • Load sufficient protein (typically 20-50 μg of total protein)

• Antibody concentration and incubation:

  • Primary antibody: Use at 1 μg/mL concentration

  • Secondary antibody: Dilute HRP-conjugated secondary antibody at 1:50,000-100,000

  • Incubate primary antibody overnight at 4°C for optimal binding

  • Wash thoroughly between antibody incubations (at least 3 x 5 min washes)

• Detection system:

  • Use enhanced chemiluminescence (ECL) systems with appropriate sensitivity

  • For low abundance proteins, consider using signal enhancement systems

• Controls:

  • Include positive control (tissue/cell line known to express ZNF583)

  • Consider using recombinant ZNF583 protein as a reference standard

How can cross-reactivity issues with ZNF583 antibodies be addressed?

Cross-reactivity can significantly impact experimental results. To address potential cross-reactivity with ZNF583 antibodies:

• Epitope analysis: Review the immunogen sequence used to generate the antibody and check for homology with other proteins, particularly other zinc finger proteins within the same family .

• Blocking peptides: Use the immunizing peptide or recombinant protein for blocking experiments to confirm specificity .

• Control tissues/cells: Test the antibody on samples known to lack ZNF583 expression to identify non-specific binding.

• Optimize blocking conditions: Increase the concentration of blocking agent (BSA or non-fat dry milk) and consider adding 0.1-0.5% Tween-20 to reduce non-specific binding.

• Antibody dilution optimization: Test a range of antibody dilutions to find the optimal signal-to-noise ratio.

• Alternative antibody selection: When possible, use monoclonal antibodies or antibodies targeting unique regions of ZNF583 to reduce cross-reactivity with related proteins.

• Species considerations: For cross-species applications, consider sequence homology (human ZNF583 shows 62% homology with mouse and 55% with rat orthologs) , which may impact antibody performance.

What is the biological significance of ZNF583 in disease contexts?

Emerging research indicates that ZNF583 may have significant implications in disease contexts, particularly in cancer:

Research into ZNF583's role in normal and disease states is still emerging, and antibody-based detection methods will be crucial for elucidating its functions and potential as a biomarker or therapeutic target.

What are the optimal conditions for immunohistochemical detection of ZNF583?

For successful immunohistochemical detection of ZNF583, consider the following methodological recommendations:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues should be sectioned at 4-6 μm thickness

  • Antigen retrieval is typically required due to formalin-induced cross-linking

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested to determine optimal conditions

• Antibody optimization:

  • Start with manufacturer's recommended dilution (typically 1:50-1:300 for IHC applications)

  • Perform a dilution series to determine optimal concentration

  • Incubate primary antibody overnight at 4°C or for 1-2 hours at room temperature

• Detection system:

  • Use appropriate secondary antibody systems (HRP or AP-based)

  • Consider signal amplification systems for low-abundance proteins

  • Optimize DAB (or other chromogen) development time to achieve optimal signal-to-noise ratio

• Controls:

  • Include positive control tissues known to express ZNF583

  • Include negative controls (primary antibody omission and isotype controls)

  • Consider using blocking peptides as additional specificity controls

• Counterstaining:

  • Use appropriate nuclear counterstains (e.g., hematoxylin) to visualize tissue architecture

  • Adjust counterstaining intensity to allow clear visualization of ZNF583 staining

How can ZNF583 expression be accurately quantified in research samples?

Accurate quantification of ZNF583 expression is essential for comparative studies. Consider these methodological approaches:

• Western blot quantification:

  • Use recombinant ZNF583 protein standards to generate a calibration curve

  • Employ digital imaging and analysis software to measure band intensity

  • Normalize to appropriate loading controls (β-actin, GAPDH, etc.)

• qRT-PCR for mRNA expression:

  • Design specific primers spanning exon-exon junctions

  • Use absolute quantification with standard curves or relative quantification with the 2^-ΔΔCt method

  • Validate qPCR efficiency and specificity before experimental use

• Immunohistochemical quantification:

  • Use digital pathology software for objective quantification

  • Score based on staining intensity and percentage of positive cells

  • Consider H-score or Allred scoring systems for semi-quantitative assessment

• Flow cytometry:

  • For intracellular staining of ZNF583, use appropriate fixation and permeabilization

  • Use isotype controls to determine background staining

  • Quantify using mean fluorescence intensity (MFI) or percentage of positive cells

• Mass spectrometry:

  • For absolute quantification, consider targeted proteomics approaches with isotope-labeled standards

  • Account for protein extraction efficiency and digestion completeness

How is ZNF583 being investigated as a potential biomarker in cancer research?

Recent findings suggest that ZNF583 has potential as a biomarker in cancer research:

When investigating ZNF583 as a biomarker, researchers should:

• Use standardized protocols for sample collection and processing

• Employ multiple detection methods (protein and mRNA level)

• Incorporate appropriate clinical data and follow-up information

• Validate findings in independent cohorts

• Consider multivariate analysis to account for confounding factors

What experimental models are suitable for studying ZNF583 function?

To investigate ZNF583 function, several experimental models can be considered:

• Cell line models:

  • Select cell lines with documented ZNF583 expression

  • Create overexpression models using cDNA expression vectors

  • Generate knockdown/knockout models using siRNA, shRNA, or CRISPR-Cas9

  • Use inducible expression systems for temporal control

• Animal models:

  • Consider species homology (62% sequence identity between human and mouse ZNF583)

  • Generate conditional knockout models to study tissue-specific effects

  • Use xenograft models to study ZNF583's role in tumor growth and metastasis

• Patient-derived samples:

  • Analyze ZNF583 expression in primary patient samples

  • Correlate expression with clinical outcomes and treatment responses

  • Consider patient-derived xenografts (PDX) for in vivo studies

• Computational approaches:

  • Use bioinformatic analysis of publicly available datasets to identify potential ZNF583 targets

  • Employ pathway analysis to understand ZNF583's role in cellular networks

  • Utilize single-cell RNA sequencing data to examine cell-type specific expression patterns