ZRANB3 Antibody, HRP conjugated

What is ZRANB3 and what cellular functions does it perform?

ZRANB3 (zinc finger, RAN-binding domain containing 3) is a 1079 amino acid protein that functions as a structure-specific ATP-dependent endonuclease . It belongs to the SNF2/RAD54 helicase family and localizes primarily in the nucleus . ZRANB3 displays a unique structure-specific endonuclease activity that allows it to cleave branched DNA structures with unusual polarity, generating an accessible 3'-OH group in the template of the leading strand .

The protein contains several functional domains including:

• One RanBP2-type zinc finger

• One helicase C-terminal domain

• One HNH domain (containing the endonuclease activity)

• One helicase ATP-binding domain

ZRANB3 plays crucial roles in:

• Maintaining genome stability at stalled replication forks

• Facilitating fork restart after replication stress

• Limiting inappropriate recombination during template switching events

• Participating in replication-associated DNA repair mechanisms

What applications is the ZRANB3 Antibody, HRP conjugated suitable for?

• Western Blotting (with recommended dilutions of 1:500-1:1000)

• Immunoprecipitation experiments for protein-protein interaction studies

• Analyzing ZRANB3 in replication stress response

It's important to note that each specific application may require optimization of antibody dilution based on your experimental system .

What is the specificity and reactivity profile of ZRANB3 antibodies?

The ZRANB3 antibody (HRP conjugated) has demonstrated reactivity with human ZRANB3 samples . The immunogen used for antibody generation is a recombinant human DNA annealing helicase and endonuclease ZRANB3 protein fragment (amino acids 370-624) .

For polyclonal antibodies like this one:

• Host species: Rabbit

• Isotype: IgG

• Species reactivity: Human

• Expected molecular weight: 123 kDa (calculated)

• Observed molecular weight: 150 kDa

The difference between calculated and observed molecular weight may be due to post-translational modifications of the protein or structural characteristics that affect migration during electrophoresis .

How can I validate the specificity of ZRANB3 antibody in experimental systems?

To properly validate ZRANB3 antibody specificity:

• Positive and negative control samples:

  • Use cell lines with known ZRANB3 expression (HEK-293 cells are positive controls)

  • Compare with ZRANB3 knockdown/knockout samples (multiple publications have demonstrated the use of ZRANB3 KD/KO systems)

• Molecular weight verification:

  • Ensure detection at the expected molecular weight of approximately 150 kDa

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be significantly reduced if antibody is specific

• Orthogonal detection methods:

  • Use different antibodies targeting distinct epitopes of ZRANB3

  • Correlate protein detection with mRNA levels via RT-PCR

• Immunoprecipitation followed by mass spectrometry:

  • Verify that the immunoprecipitated protein is indeed ZRANB3

What experimental protocols are recommended for detecting ZRANB3-protein interactions?

ZRANB3 forms important interactions with several key proteins in DNA replication and repair pathways. To study these interactions:

• Co-immunoprecipitation (Co-IP):

  • Use anti-ZRANB3 antibody to pull down protein complexes

  • Western blot for interaction partners such as:

    • PCNA (proliferating cell nuclear antigen)

    • RNR-α (ribonucleotide reductase alpha)

    • MCM subunits (minichromosome maintenance protein complex)

• Proximity ligation assay (PLA):

  • For visualizing protein interactions in situ

  • Particularly useful for examining ZRANB3-PCNA interactions at replication factories

• Yeast two-hybrid verification:

  • The ZRANB3 C-terminus (residues 929-1079) was identified as an RNR-α-specific interactor in Y2H screens

  • Can be used to verify specific domain interactions

• In vitro binding assays:

  • Using purified recombinant proteins

  • Previous research showed recombinantly-purified C-terminal domains of ZRANB3 (aa 929-1079, containing HNH-domain and APIM-motif) directly bind to full-length recombinant RNR-α in vitro

• Competition experiments:

  • To evaluate binding dynamics between ZRANB3 and competing partners

  • Research has shown PCNA can interrupt pre-established ZRANB3-RNR-α interactions

How can I optimize detection of ZRANB3 in subcellular fractionation experiments?

ZRANB3 functions in the nucleus, particularly at replication foci, making subcellular fractionation an important approach:

• Fractionation protocol optimization:

  • Use gentle lysis conditions to preserve protein-protein interactions

  • Include phosphatase inhibitors to maintain post-translational modifications

  • Verify fraction purity using markers: PCNA (nuclear), GAPDH (cytoplasmic)

• Visualization techniques:

  • Fluorescence microscopy shows ZRANB3 in patterned nuclear signals resembling replication foci

  • Co-staining with BrdU and PCNA confirms localization to sites of active DNA synthesis

• Extraction considerations:

  • ZRANB3 associates with chromatin, requiring proper extraction conditions

  • Include DNase treatment if necessary to release chromatin-bound fractions

  • Buffer compositions significantly affect extraction efficiency

• Analysis parameters:

  • Quantify nuclear vs. cytoplasmic distribution

  • Evaluate co-localization with replication markers

  • Assess changes in localization following DNA damage or replication stress

What experimental approaches can detect ZRANB3 activity in replication stress response?

To study ZRANB3's function in replication stress response:

• DNA damage induction protocols:

  • Treatment with mitomycin-C has been shown to recruit ZRANB3 to small PCNA foci

  • Other replication stress inducers like hydroxyurea or aphidicolin can be used

• DNA fiber analysis:

  • Crucial method to assess ZRANB3's role in DNA replication dynamics

  • ZRANB3 deficiency (70% knockdown) results in ~30% decrease in DNA synthesis rate by fiber-spreading and fiber-combing techniques

  • EdU/BrdU dual-pulse labeling also reveals 30-40% suppression of BrdU incorporation upon ZRANB3 siRNA knockdown

• Replication fork restart assays:

  • Measure recovery of DNA synthesis after transient replication stress

  • ZRANB3 is required for efficient restart of stalled replication forks

• Structure-specific endonuclease activity assays:

  • ZRANB3 exhibits ATP-dependent endonuclease activity via its C-terminal HNH domain

  • Test cleavage of branched DNA structures with specific polarity

• Interaction dynamics:

  • Evaluate how ZRANB3-PCNA interaction changes during normal replication versus stress conditions

  • Assess competition between RNR-α and PCNA for ZRANB3 binding

What are common issues with ZRANB3 antibody detection and how can they be resolved?

How should experiments be designed to study the ZRANB3-RNR-α-PCNA interaction axis?

Based on published research showing competitive binding between RNR-α and PCNA for ZRANB3 , consider these experimental approaches:

• Competition assays:

  • Co-IP experiments have shown that overexpression of RNR-α-NLS reduces ZRANB3-PCNA association

  • In vitro binding experiments demonstrated that excess PCNA reduces ZRANB3(HNH-APIM)-RNR-α direct interaction

  • Recombinant PCNA can fully elute pre-bound RNR-α from ZRANB3(HNH-APIM)-coated beads within 30 minutes

• Domain-specific interaction mapping:

  • The C-terminus of ZRANB3 (residues 929-1079) containing the HNH domain and APIM motif is crucial for RNR-α interaction

  • The APIM motif (aa 1074-1078) is required for ZRANB3-RNR-α interaction

  • ZRANB3 also contains a PIP-box (aa 519-526) for PCNA interaction, though this is not required for RNR-α binding

• Localization studies:

  • RNR-α-NLS overexpression reduces ZRANB3-PCNA puncta co-localization

  • Upon DNA damage (mitomycin-C treatment), ZRANB3 is recruited to small PCNA foci that do not contain RNR-α

• Functional consequences:

  • Test DNA synthesis rates in contexts where the balance between these interactions is altered

  • Evaluate effects on replication fork stability and restart efficiency

What controls are essential when using ZRANB3 antibodies in immunofluorescence studies?

For reliable immunofluorescence studies with ZRANB3 antibodies:

• Essential negative controls:

  • Primary antibody omission

  • ZRANB3 knockdown/knockout cells

  • Isotype control (rabbit IgG)

• Positive controls:

  • Cells with known ZRANB3 expression (HEK-293 works well)

  • YFP-ZRANB3 overexpressing cells as reference pattern

• Localization verification:

  • Co-staining with replication markers:

    • PCNA (shows colocalization with ZRANB3)

    • BrdU pulse-labeling (ZRANB3 overlaps with sites of BrdU incorporation)

  • Nuclear counterstaining (DAPI)

• Functional contexts:

  • Untreated vs. DNA damage-induced conditions

  • Different cell cycle phases

  • Replication stress induction

How can ZRANB3 antibodies be used to study replication fork dynamics?

ZRANB3 antibodies can provide valuable insights into replication fork dynamics:

• Chromatin immunoprecipitation (ChIP):

  • To detect ZRANB3 recruitment to replication forks

  • ChIP-seq can map genome-wide binding sites

• iPOND (isolation of Proteins On Nascent DNA):

  • Combine with ZRANB3 antibodies to study protein associations at active replication forks

  • Compare normal versus stressed replication conditions

• DNA combing with immunodetection:

  • Measure fork progression rates

  • Research has shown ZRANB3 deficiency causes ~30% decrease in DNA synthesis rate

• Cell cycle synchronization experiments:

  • Analyze ZRANB3 dynamics throughout S-phase

  • Compare early versus late replication timing

• DNA damage response studies:

  • Assess ZRANB3 recruitment kinetics following various DNA damaging agents

  • Compare with recruitment of other replication stress response factors

What techniques can detect ZRANB3 structure-specific endonuclease activity?

To study the unique endonuclease activity of ZRANB3:

• In vitro enzymatic assays:

  • Using purified recombinant ZRANB3

  • Synthetic branched DNA substrates with fluorescent labels

  • Analyze cleavage products by gel electrophoresis

• Activity requirements validation:

  • ATP dependence (ZRANB3 endonuclease activity is coupled to ATP hydrolysis)

  • Structure specificity (ZRANB3 cleaves branched DNA with unusual polarity)

  • Test mutants (particularly in the HNH domain) to confirm specific residues required for activity

• Cellular assays:

  • Use catalytically inactive ZRANB3 mutants as dominant negatives

  • Compare phenotypes with ZRANB3 knockdown

• Substrate preference characterization:

  • Test various DNA structures (replication forks, D-loops, Holliday junctions)

  • Determine optimal conditions for enzymatic activity

How can researchers integrate ZRANB3 studies with broader genome stability research?

ZRANB3's role in genome stability can be studied in these integrated contexts:

• Genomic instability phenotypes:

  • Micronuclei formation assays

  • Sister chromatid exchange frequency

  • Chromosomal aberration analysis

  • DNA break accumulation (γH2AX foci)

• Integration with DNA repair pathways:

  • Study interactions between ZRANB3 and:

    • Homologous recombination factors

    • Translesion synthesis machinery

    • Nucleotide excision repair components

• Cancer relevance studies:

  • Analyze ZRANB3 expression/mutations in cancer datasets

  • Evaluate synthetic lethality with other genome stability genes

  • Test sensitization to chemotherapeutic agents

• Multi-omics approaches:

  • Combine proteomics, genomics, and functional assays

  • Map the ZRANB3 interactome under various conditions

  • Identify novel regulatory mechanisms

How should researchers interpret discrepancies in ZRANB3 molecular weight detection?

ZRANB3 has a calculated molecular weight of 123 kDa based on its 1079 amino acid sequence, but is commonly observed at approximately 150 kDa in Western blot analyses . Researchers should consider:

How can researchers analyze competition between PCNA and RNR-α for ZRANB3 binding?

Based on the "competition model" described in research , where nuclear RNR-α forms a complex with ZRANB3, preventing ZRANB3-PCNA interaction in the absence of DNA damage:

• Quantitative binding analysis:

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • FRET-based assays for real-time interaction monitoring

• Data visualization techniques:

  • Plot ZRANB3-PCNA association versus RNR-α concentration

  • Create competition curves with varying ratios of proteins

  • Use statistical modeling to determine binding constants

• Experimental evidence interpretation:

  • Overexpression of RNR-α-NLS reduces ZRANB3-PCNA association in co-IP experiments

  • Excess PCNA can reduce ZRANB3-RNR-α direct interaction in vitro

  • PCNA can fully elute pre-bound RNR-α from ZRANB3 within 30 minutes

  • ZRANB3 and nuclear RNR-α partially co-localize independently of reductase activity

• Kinetic considerations:

  • Time-course experiments to determine association/dissociation rates

  • Protein concentration effects on equilibrium shifting

  • DNA damage-induced changes in binding preferences