zranb1b Antibody

What is ZRANB1B and what are its primary functions?

ZRANB1B (Zinc finger RAN-binding domain-containing protein 1-B) functions as a ubiquitin thioesterase that specifically hydrolyzes 'Lys-29'-linked and 'Lys-33'-linked diubiquitin. It plays multiple critical roles in cellular function:

• Acts as a deubiquitinase that specifically removes ubiquitin moieties from polyubiquitinated substrates, influencing their stability and degradation

• Functions as a positive regulator of the Wnt signaling pathway by deubiquitinating APC protein

• Regulates autophagy through deubiquitination of PIK3C3/VPS34, promoting autophagosome maturation

• Influences cell morphology, cytoskeletal organization, stress fiber dynamics, and cell migration

• Serves as an important regulator of protein quality control and cellular function

What species reactivity is confirmed for commercial ZRANB1B antibodies?

Based on available research and product information, ZRANB1B antibodies have been confirmed to react with:

• Zebrafish ZRANB1B: The rabbit polyclonal antibody is specifically designed for this target and has been validated in Western blot applications

• Human ZRANB1: The goat polyclonal antibody (PAB11595) has been validated for detection of human ZRANB1 in Western blot and ELISA applications

For zebrafish studies, the immunogen corresponds to a recombinant fragment protein within amino acids 100-350 of zebrafish ZRANB1B .

What applications are ZRANB1B antibodies validated for?

Commercial ZRANB1B antibodies have been validated for several research applications:

• Western Blot: Both rabbit and goat polyclonal antibodies are suitable, with recommended dilutions of 1-3 μg/mL

• ELISA: The goat polyclonal antibody is recommended at a dilution of 1:4000

• Immunohistochemistry: ZRANB1 expression has been successfully analyzed in tissue samples using immunohistochemical staining

The optimal working dilution should be determined by the end user for specific experimental conditions and sample types .

What is the theoretical molecular weight of ZRANB1 and how does this affect antibody applications?

The theoretical molecular weight of human ZRANB1 is 81 kDa according to product specifications . This information is critical for researchers when:

• Interpreting Western blot results: The main band should appear around 81 kDa

• Developing appropriate gel separation parameters

• Assessing antibody specificity: Presence of bands at significantly different molecular weights may indicate non-specific binding or protein degradation

• Optimizing sample preparation protocols to preserve the intact protein

How does ZRANB1 expression correlate with cancer progression and clinical outcomes?

Research has revealed significant correlations between ZRANB1 expression and cancer progression:

These findings suggest antibodies targeting ZRANB1/ZRANB1B are valuable tools for cancer research, prognostic studies, and potential therapeutic development.

What is the molecular mechanism by which ZRANB1 promotes cancer pathogenesis?

ZRANB1 promotes cancer progression through several molecular mechanisms:

• It functions as a specific deubiquitinase for EZH2, a key epigenetic regulator

• ZRANB1 physically binds, deubiquitinates, and stabilizes EZH2 protein, preventing its degradation

• It specifically removes K33-linked polyubiquitin chains from EZH2, as demonstrated through in vitro deubiquitination assays

• Depletion of ZRANB1 in breast cancer cells results in significant EZH2 destabilization (reduced by 60-70%) and growth inhibition

• ZRANB1 knockout abrogates H3K27 trimethylation but not H3K27me1 or H3K27me2, affecting epigenetic regulation

• ZRANB1 knockout also decreases protein levels of other PRC2 components including SUZ12 and EED

These findings position ZRANB1 as a critical upstream regulator of cancer-promoting epigenetic mechanisms.

What experimental approaches can be used to study ZRANB1B's deubiquitinase activity?

Several sophisticated experimental approaches can be employed to study ZRANB1B's deubiquitinase activity:

• In vivo ubiquitination assays: Compare polyubiquitination of target proteins (e.g., EZH2) between ZRANB1-expressing and ZRANB1-knockout cells under denaturing conditions

• In vitro deubiquitination assays: Incubate purified ZRANB1 with ubiquitinated substrate proteins in a cell-free system to directly assess enzymatic activity

• Linkage-specific ubiquitin analysis: Use ubiquitin mutants (K33, etc.) to determine ZRANB1's specificity for different ubiquitin chain linkages

• Domain mapping: Generate truncation mutants (e.g., NZFs, NZFs+AnkUBD, AnkUBD+OTU) to identify domains required for substrate interaction and deubiquitination

• Cellular localization studies: Employ immunofluorescence with ZRANB1B antibodies to determine subcellular localization and co-localization with substrates

These approaches provide complementary data on ZRANB1B's enzymatic function, substrate specificity, and regulatory mechanisms.

What structural domains of ZRANB1 are important for its function and antibody recognition?

ZRANB1 consists of several functional domains that are critical for its deubiquitinase activity:

• Three N-terminal zinc-finger (NZF) domains: Likely involved in protein-protein interactions

• A central ankyrin repeat ubiquitin-binding domain (AnkUBD): Mediates binding to ubiquitinated substrates

• A C-terminal OTU (ovarian tumor) domain: Essential for deubiquitinase catalytic activity

Experimental evidence demonstrates that:

• The OTU domain mediates physical interaction with substrates like EZH2

• The OTU domain is required for the stabilization of EZH2 by ZRANB1

• ZRANB1 exhibits both nuclear and cytoplasmic expression, with co-localization with EZH2 observed in the nucleus

For antibody development and recognition, these structural considerations affect epitope selection, with the zebrafish ZRANB1B antibody targeting a region within amino acids 100-350 .

What are the recommended protocols for Western blot detection of ZRANB1B?

Based on validated methodologies, the following protocol parameters are recommended for Western blot detection of ZRANB1B:

• Sample preparation: RIPA buffer is effective for protein extraction from tissues and cell lines

• Protein loading: 35 μg protein per lane has been successfully used for detection in human cerebellum lysate

• Antibody dilution:

  • Goat polyclonal antibody: 1-3 μg/mL is the recommended range, with 0.1 μg/mL successfully used in published experiments

  • Rabbit polyclonal antibody: Follow manufacturer's recommendation for the specific lot

• Primary antibody incubation: 1 hour at room temperature has been demonstrated to be effective

• Detection method: Chemiluminescence provides sufficient sensitivity for ZRANB1B detection

• Expected molecular weight: Approximately 81 kDa for human ZRANB1

Optimization of these parameters may be necessary depending on the specific sample type and experimental conditions.

How should ZRANB1B antibodies be stored and handled for optimal performance?

Proper storage and handling of ZRANB1B antibodies is critical for maintaining their performance:

• Storage temperature: Store at -20°C for long-term stability

• Buffer composition: Typically provided in Tris saline, pH 7.3 with 0.5% BSA and 0.02% sodium azide

• Aliquoting recommendation: Prepare small aliquots to avoid repeated freeze-thaw cycles which can degrade antibody performance

• Concentration: Antibodies are typically provided at 0.5 mg/mL

• Safety precautions: Products contain sodium azide, which is hazardous and should be handled by trained personnel only

• Shipping conditions: Antibodies are generally shipped with cold packs and should be transferred to -20°C storage immediately upon receipt

Following these guidelines will help ensure consistent antibody performance across experiments.

What controls should be included when validating ZRANB1B antibody specificity?

Comprehensive validation of ZRANB1B antibodies should include multiple controls:

• Positive control tissues/cells: HCC cell lines (Huh7, MHCC97H, HepG2, HCCLM3) show high ZRANB1 expression and can serve as positive controls

• Negative controls: Normal hepatocyte lines (HL7702, THLE-3) show lower ZRANB1 expression , while ZRANB1 knockout cells generated via CRISPR-Cas9 provide definitive negative controls

• Peptide competition assay: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining

• Correlation with mRNA levels: Compare protein detection with quantitative RT-PCR results for ZRANB1B expression

• Multiple antibody validation: Use different antibodies targeting distinct ZRANB1B epitopes to confirm specificity

This multi-layered approach to validation ensures that experimental findings accurately reflect ZRANB1B biology.

What factors can affect ZRANB1B detection in experimental samples?

Several technical factors can influence ZRANB1B detection:

• Expression levels: ZRANB1 expression varies significantly between normal and cancer tissues, requiring optimization of detection protocols for different sample types

• Subcellular localization: ZRANB1 exhibits both nuclear and cytoplasmic localization, which may affect extraction efficiency depending on the lysis method

• Post-translational modifications: As a protein involved in the ubiquitin system, ZRANB1 itself may undergo modifications that affect antibody recognition

• Protein-protein interactions: ZRANB1's interaction with binding partners may mask epitopes in certain experimental contexts

• Sample preparation: Inadequate lysis or protein degradation during sample preparation can significantly impact detection

Understanding these factors is essential for troubleshooting inconsistent results and optimizing experimental protocols.

How can ZRANB1B antibodies be used in cancer biomarker research?

ZRANB1B antibodies offer valuable applications in cancer biomarker research:

• Tissue microarray analysis: Immunohistochemical staining of large patient cohorts to assess ZRANB1 expression across cancer types and stages

• Prognostic evaluation: Correlation of ZRANB1 expression levels with patient outcomes through Kaplan-Meier survival analysis

• Comparative tissue analysis: Quantitative comparison of ZRANB1 expression between tumor and adjacent normal tissues

• Clinicopathological correlation: Assessment of ZRANB1 expression in relation to tumor parameters (size, stage, invasion, metastasis)

• Western blot quantification: Precise measurement of ZRANB1 protein levels in patient-derived samples

These applications can help establish ZRANB1 as a clinically relevant biomarker for cancer diagnosis and prognosis.

What experimental designs can assess ZRANB1B's role in the ubiquitin-proteasome system?

To investigate ZRANB1B's function in the ubiquitin-proteasome system, researchers can implement these experimental designs:

• Substrate identification studies: Immunoprecipitate ZRANB1B followed by mass spectrometry to identify novel substrate proteins

• In vitro deubiquitination assays: Examine ZRANB1B's ability to remove specific ubiquitin linkages from purified substrates

• Chain-specificity analysis: Use linkage-specific ubiquitin mutants to determine ZRANB1B's preference for different ubiquitin chain types

• Proteasome inhibition studies: Combine ZRANB1B manipulation with proteasome inhibitors to assess effects on substrate degradation

• Domain mapping: Generate ZRANB1B truncation mutants to identify regions required for substrate recognition and deubiquitination

For example, the following experimental workflow has proven effective:

• Transfect cells with control or ZRANB1-targeting siRNAs

• Assess changes in target protein (e.g., EZH2) polyubiquitination under denaturing conditions

• Measure target protein levels and stability

• Evaluate functional consequences through phenotypic assays

How can ZRANB1B antibodies be used in developmental biology research?

The availability of zebrafish-specific ZRANB1B antibodies enables several applications in developmental biology:

• Temporal expression analysis: Track ZRANB1B expression during different developmental stages using Western blot and immunohistochemistry

• Tissue-specific expression patterns: Map ZRANB1B distribution across developing tissues

• Wnt signaling studies: Investigate ZRANB1B's role as a positive regulator of Wnt-induced transcription during development

• Functional studies: Combine antibody detection with genetic manipulation of zranb1b (morpholino knockdown or CRISPR knockout)

• Cytoskeletal organization: Examine ZRANB1B's role in regulating cell morphology and migration during embryogenesis

These approaches can illuminate ZRANB1B's contributions to normal development and provide context for its dysregulation in pathological conditions.

What considerations are important when using ZRANB1B antibodies for co-immunoprecipitation experiments?

Co-immunoprecipitation with ZRANB1B antibodies requires careful consideration of several factors:

• Antibody suitability: Confirm the antibody can recognize native (non-denatured) ZRANB1B and doesn't target interaction interfaces

• Epitope accessibility: The OTU domain of ZRANB1 mediates interactions with proteins like EZH2, so antibodies targeting this region may interfere with certain protein-protein interactions

• Buffer conditions: Use lysis buffers that preserve protein-protein interactions while efficiently extracting ZRANB1B from its subcellular compartments

• Pre-clearing samples: Implement thorough pre-clearing to reduce non-specific binding

• Controls: Include IgG controls, input samples, and where possible, ZRANB1-knockout samples as negative controls

Successful co-immunoprecipitation has demonstrated ZRANB1's interaction with EZH2 and other proteins, providing valuable insights into its functional network .

How does ZRANB1B research contribute to cancer therapeutic development?

Research using ZRANB1B antibodies has revealed promising therapeutic opportunities:

• EZH2 stabilization: ZRANB1 functions as an EZH2 deubiquitinase, suggesting that ZRANB1 inhibition could destabilize EZH2 in cancers where EZH2 enzymatic inhibitors are ineffective

• TNBC treatment: Systemic delivery of ZRANB1 siRNA showed marked antitumor and antimetastatic effects in preclinical models of triple-negative breast cancer

• Small-molecule inhibitors: A ZRANB1 inhibitor has been shown to destabilize EZH2 and inhibit the viability of TNBC cells

• Biomarker development: ZRANB1 levels correlate with poor survival in breast cancer patients, suggesting utility as a prognostic marker

• Hepatocellular carcinoma: ZRANB1 overexpression is associated with poor prognosis in HCC, suggesting it as a candidate prognostic biomarker for treatment stratification

These findings position ZRANB1 as a promising target for therapeutic intervention in multiple cancer types.

What are the advantages of targeting ZRANB1B compared to direct EZH2 inhibition?

Targeting ZRANB1B offers several potential advantages over direct EZH2 inhibition:

• Broader efficacy: Many cancers do not respond to EZH2 enzymatic inhibitors because EZH2 can promote cancer independently of its histone methyltransferase activity

• Dual mechanism: ZRANB1 inhibition destabilizes both EZH2 and other PRC2 components (SUZ12, EED), potentially providing more comprehensive suppression of PRC2 function

• Alternative to resistance: Cancers that develop resistance to direct EZH2 inhibitors might remain sensitive to ZRANB1B-targeted approaches

• Multiple downstream effects: ZRANB1B regulates multiple substrates beyond EZH2, potentially addressing multiple cancer pathways simultaneously

• Preservation of normal function: Targeted reduction of ZRANB1B may selectively affect cancer cells that overexpress this protein while sparing normal cells

These comparative advantages make ZRANB1B an attractive alternative target for therapeutic development.