znf598 Antibody

What is ZNF598 and why is it important in molecular biology research?

ZNF598 (Zinc Finger Protein 598) functions as an E3 ubiquitin ligase that plays a critical role in ribosome-associated quality control (RQC). This pathway activates when ribosomes stall during translation, particularly at poly(A) sequences. ZNF598 acts as a ribosome collision sensor that recognizes and binds collided di-ribosomes, which arise when a trailing ribosome encounters a slower leading ribosome . Upon binding to colliding ribosomes, ZNF598 mediates monoubiquitination of 40S ribosomal proteins RPS10/eS10 and RPS3/uS3, and 'Lys-63'-linked polyubiquitination of RPS20/uS10 . This function is critical for preventing the synthesis of potentially harmful protein products, making ZNF598 an important research target for understanding translational quality control mechanisms.

How do I select the appropriate ZNF598 antibody for my research application?

Selection should be based on:

• Validated applications: Confirm the antibody has been validated for your specific application (Western blot, immunohistochemistry, immunofluorescence)

• Species reactivity: Verify compatibility with your experimental model (human, mouse, rat)

• Antibody type:

  • Polyclonal antibodies: Offer higher sensitivity but potentially lower specificity

  • Monoclonal antibodies: Provide higher specificity and lot-to-lot consistency

• Immunogen information: Check if the epitope is in a functionally relevant domain of ZNF598

• Validation data: Review published validation data showing specificity in knockout models

For functional studies of the E3 ligase activity, select antibodies raised against epitopes outside the RING domain to avoid interference with protein function .

What are the structural features of ZNF598 relevant to antibody selection?

ZNF598 is a 904 amino acid protein containing:

• One N-terminal RING domain characteristic of E3 ubiquitin ligases

• Four N-terminal C2H2-type zinc finger motifs

• One C-terminal C2H2-type zinc finger motif

When studying specific functions of ZNF598, select antibodies that recognize epitopes in regions that won't interfere with the domain of interest .

What are the optimal conditions for using ZNF598 antibodies in Western blot applications?

For optimal Western blot results with ZNF598 antibodies:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors

  • For ribosome-associated ZNF598, consider using polysome fractionation methods

• Recommended dilutions:

  • Polyclonal antibodies: 1:500-1:3000

  • Monoclonal antibodies: 1:2000 for cellular lysates

• Detection considerations:

  • Expected molecular weight: ~99 kDa

  • Use positive controls: HEK293 or HCT116 cell lysates show good endogenous expression

  • Negative controls: ZNF598 knockout cell lysates are ideal for validation

• Blocking conditions:

  • 5% non-fat dry milk or BSA in TBST (depending on the specific antibody)

For detecting ubiquitinated ribosomal protein substrates of ZNF598, consider using phosphatase inhibitors and deubiquitinase inhibitors in your lysis buffer to preserve post-translational modifications .

How can ZNF598 antibodies be optimized for immunofluorescence and immunocytochemistry applications?

For successful IF/ICC applications with ZNF598 antibodies:

• Fixation methods:

  • 4% paraformaldehyde (PFA) for 10-15 minutes at room temperature

  • Avoid methanol fixation which can disrupt protein epitopes

• Permeabilization:

  • 0.1-0.5% Triton X-100 for 5-10 minutes

  • Alternative: 0.1% saponin for gentler permeabilization

• Antibody dilutions:

  • Typical range: 1:100-1:1000 for both polyclonal and monoclonal antibodies

  • Validate optimal dilution for each new lot

• Controls and validation:

  • Positive control: HeLa, A431, or HEK293 cells show detectable endogenous expression

  • Negative control: ZNF598 knockout cells or siRNA-treated cells

  • Co-localization markers: Ribosomal proteins for validation of association with translating ribosomes

• Signal enhancement techniques:

  • Tyramide signal amplification if endogenous levels are low

  • Extended primary antibody incubation (overnight at 4°C)

Note that endogenous ZNF598 typically shows cytoplasmic distribution and does not relocalize to stress granules under arsenite-induced stress conditions, unlike many RNA-binding proteins .

What controls should be included when validating ZNF598 antibodies for research applications?

A comprehensive validation approach should include:

• Positive controls:

  • Cell lines with known ZNF598 expression (HEK293, U2OS, HCT116)

  • Overexpression systems with tagged ZNF598 for antibody calibration

• Negative controls:

  • ZNF598 knockout cell lines generated by CRISPR/Cas9

  • siRNA or shRNA knockdown samples showing reduced signal

  • Peptide competition assays to demonstrate specificity

• Application-specific controls:

  • For Western blot: Size markers to confirm the expected 99 kDa band

  • For IP: IgG control to assess non-specific binding

  • For IHC/ICC: Secondary antibody-only controls to evaluate background

• Functional validation:

  • Correlate antibody detection with functional readouts (e.g., polysome profiles in ZNF598-OE vs. ZNF598-KO cells)

  • Detection of ubiquitinated ribosomal proteins (RPS10, RPS3, RPS20) in wild-type vs. ZNF598 knockout conditions

This validation strategy ensures that antibody-based detection accurately reflects both the presence and functional status of ZNF598 in experimental systems .

How can ZNF598 antibodies be utilized to study ribosome collision and quality control mechanisms?

ZNF598 antibodies can be leveraged for multiple advanced applications in ribosome quality control research:

• Co-immunoprecipitation (Co-IP) of collision complexes:

  • Use ZNF598 antibodies to pull down native collision complexes

  • Analyze associated components by mass spectrometry to identify novel RQC factors

  • Compare bound RNAs before and after ribosome stalling induction

• Proximity labeling approaches:

  • Combine with BioID or APEX2 proximity labeling to identify proteins in close proximity to ZNF598 during RQC events

  • Map the dynamic composition of collision complexes under different cellular stresses

• Ribosome profiling analysis:

  • Use ZNF598 antibodies for ribosome profiling to map ZNF598 association with specific mRNA regions

  • Compare with polyA tract distribution to correlate with stalling sites

• Ubiquitination site mapping:

  • Detect and quantify the ZNF598-dependent ubiquitination of ribosomal proteins

  • Monitor changes in RPS10, RPS3, and RPS20 ubiquitination under various conditions

• Live-cell imaging of RQC events:

  • Combine with fluorescently-tagged ribosomal proteins to visualize collision events

  • Track the kinetics of ZNF598 recruitment to stalled ribosomes

These approaches can reveal new insights into the molecular mechanisms of ribosome-associated quality control and the central role of ZNF598 in this process .

What are the methodological considerations when using ZNF598 antibodies to investigate RNA-protein interactions?

When investigating ZNF598's RNA-binding properties:

• RNA immunoprecipitation (RIP) protocols:

  • Use formaldehyde crosslinking (0.1-0.3%) to preserve transient RNA-protein interactions

  • Include RNase inhibitors throughout lysis and immunoprecipitation

  • Validate with known RNA targets or through PAR-CLIP results

• PAR-CLIP considerations:

  • PAR-CLIP has revealed ZNF598 cross-links to tRNAs, mRNAs, and rRNAs

  • Special attention to tRNA^Lys(UUU) which shows 10-fold enrichment in cross-linking

  • Include controls for non-specific RNA binding

• Analysis of RNA specificity:

  • ZNF598 shows preference for AAA codons/poly(A) sequences

  • Design experiments to compare binding affinity for different RNA sequences

  • Consider competitive binding assays with synthetic RNA oligos

• Zinc finger domain mutation controls:

  • Include C2H2 zinc finger mutants to assess the contribution of these domains to RNA binding

  • Compare wild-type and mutant ZNF598 in RNA-binding assays

• In vitro binding assays:

  • Purified recombinant ZNF598 can be used for direct RNA binding studies

  • EMSA (Electrophoretic Mobility Shift Assay) with labeled RNA probes

  • Filter binding assays to quantify affinity constants

These approaches can help delineate the molecular basis for ZNF598's role in recognizing specific RNA features that trigger ribosome-associated quality control .

How can ZNF598 antibodies be employed to study the relationship between ribosome stalling and neurodegenerative diseases?

Emerging research suggests connections between ribosome quality control and neurodegenerative disorders:

• Patient-derived cell analysis:

  • Use ZNF598 antibodies to compare RQC component levels and localization in cells from patients vs. healthy controls

  • Analyze post-mortem brain tissue for ZNF598 expression patterns and co-localization with disease markers

• Stress response studies:

  • Monitor ZNF598-dependent ubiquitination during cellular stress conditions relevant to neurodegeneration

  • Quantify changes in RPS10/RPS20 ubiquitination in disease models using modification-specific antibodies

• Protein aggregation models:

  • Investigate whether ZNF598 co-localizes with protein aggregates in neurodegenerative disease models

  • Analyze the effect of ZNF598 manipulation on aggregate formation using immunofluorescence

• Translation fidelity assessment:

  • Use reporter constructs with disease-associated repeat expansions

  • Compare ZNF598 recruitment and RQC activity between normal and disease conditions

• Neuronal-specific RQC analysis:

  • Employ immunohistochemistry to map ZNF598 expression in different neuronal populations

  • Correlate with vulnerability patterns in neurodegenerative diseases

This research direction may reveal how defects in ribosome-associated quality control contribute to protein homeostasis disruption in neurodegenerative disorders .

How can researchers troubleshoot non-specific binding or poor signal when using ZNF598 antibodies?

When encountering issues with ZNF598 antibody performance:

• Non-specific binding in Western blot:

  • Increase blocking time/concentration (5-10% blocking agent)

  • Use more stringent washing conditions (0.1-0.3% Tween-20)

  • Test alternative blocking agents (milk vs. BSA)

  • Validate with ZNF598 knockout samples to identify true specific bands

• Weak signal detection:

  • Optimize primary antibody concentration and incubation time

  • Try different antibody clones or lots

  • Consider signal amplification methods

  • Enrich for ZNF598 through subcellular fractionation or polysome isolation

• Background in immunofluorescence:

  • Optimize fixation protocol (PFA concentration and time)

  • Test different permeabilization agents and conditions

  • Include appropriate blocking of Fc receptors

  • Use Sudan Black to reduce autofluorescence in tissue sections

• Cross-reactivity assessment:

  • Check species homology of the immunogen sequence

  • Perform peptide competition assays

  • Compare multiple antibodies targeting different epitopes

These troubleshooting approaches can help researchers optimize ZNF598 antibody performance across different experimental systems and applications .

How do researchers interpret ZNF598 antibody results in the context of ribosome quality control studies?

Proper interpretation of ZNF598 antibody data requires:

• Expression level analysis:

  • ZNF598 is expressed at sub-stoichiometric levels compared to ribosomes

  • Changes in expression may indicate cellular stress responses

  • Compare across tissue/cell types to identify context-specific regulation

• Localization pattern interpretation:

  • Cytoplasmic distribution is expected (association with translating ribosomes)

  • Unlike many RNA-binding proteins, ZNF598 does not relocalize to stress granules under arsenite-induced stress

  • Alterations in this pattern may indicate disruption of normal function

• Polysome profile correlation:

  • ZNF598 overexpression shifts polysome profiles toward monosomes (translational repression)

  • ZNF598 knockout shifts toward heavier polysomes

  • Western blot analysis of polysome fractions should show ZNF598 association with heavy polysomes

• Ubiquitination status assessment:

  • ZNF598-dependent ubiquitination of RPS10 (K138/K139) is a key functional readout

  • Compare wild-type vs. C29A RING domain mutant effects

  • Use ubiquitin-specific antibodies to detect modified ribosomal proteins

• Functional readouts:

  • Reporter assays with stall sequences (e.g., poly(A) tracts)

  • Assess readthrough vs. stalling in ZNF598 manipulated conditions

  • Correlate with ubiquitination status of target ribosomal proteins

Context-specific interpretation helps distinguish between direct ZNF598 effects and secondary consequences in complex experimental systems .

What are the most common sources of data misinterpretation when using ZNF598 antibodies, and how can they be avoided?

Researchers should be aware of these potential pitfalls:

• Redundancy in RQC pathways:

  • ZNF598 knockout may show partial phenotypes due to compensatory mechanisms

  • Use acute depletion (e.g., auxin-inducible degron) to minimize adaptation

  • Compare genetic knockout with antibody-based detection to identify discrepancies

• Post-translational modification effects:

  • ZNF598 itself may be subject to regulation by PTMs

  • Changes in antibody detection may reflect modifications rather than expression changes

  • Use multiple antibodies targeting different epitopes for validation

• E3 ligase activity vs. presence:

  • Antibody detection confirms presence but not necessarily activity

  • C29A mutant appears similar to wild-type in expression assays but lacks function

  • Combine with functional ubiquitination assays for complete interpretation

• Species-specific considerations:

  • Despite high conservation (human ZNF598 shares 95% sequence identity with rat), functional differences may exist

  • Validate antibody specificity and function separately for each species model

  • Consider species-specific immunogens when available

• Technical artifacts in methodology:

  • Overexpression can create artificial associations and phenotypes

  • Fixation methods can affect epitope accessibility and apparent localization

  • Ribosome profiling artifact can arise due to ZNF598's RNA-binding properties

By anticipating these common misinterpretation sources, researchers can design more robust experiments with appropriate controls and validation strategies .

How are ZNF598 antibodies being used to investigate novel functions beyond canonical ribosome quality control?

Recent research is expanding our understanding of ZNF598 functions:

• Viral infection responses:

  • ZNF598 is required for poxvirus protein synthesis

  • Mediates ubiquitination of ribosomal proteins during viral infection

  • May act as a negative regulator of interferon-stimulated gene expression

  • Antibodies can help track ZNF598 redistribution during infection

• mRNA regulation beyond stalling:

  • Functions as an adapter recruiting the 4EHP-GYF2 complex to mRNAs

  • Potential roles in translational regulation independent of ubiquitination activity

  • Antibodies can help identify novel mRNA targets via RIP-seq approaches

• Specialized ribosome hypothesis:

  • Investigating whether ZNF598 marks specialized ribosomes for specific mRNAs

  • Comparing ZNF598 association with different ribosome populations

  • Using antibodies to isolate and characterize potentially specialized ribosomes

• Cancer biology connections:

  • Examining ZNF598 expression and localization changes in various cancer types

  • Investigating correlations between ZNF598 activity and cancer cell stress responses

  • Using tissue microarrays with ZNF598 antibodies for expression profiling across tumor samples

These emerging research directions highlight the expanding role of ZNF598 beyond its canonical function in ribosome-associated quality control .

What methodological advances are improving the application of ZNF598 antibodies in ribosome dynamics research?

Cutting-edge methodological approaches include:

• Super-resolution microscopy applications:

  • STORM or PALM microscopy to visualize individual ribosome collision events

  • Tracking ZNF598 recruitment to ribosomes with nanometer precision

  • Requires highly specific antibodies or fusion proteins for accurate localization

• Cryo-EM structural studies:

  • Using antibodies to stabilize ZNF598-ribosome complexes for structural determination

  • Fab fragments as fiducial markers for single-particle reconstruction

  • Mapping the precise binding interface between ZNF598 and collided ribosomes

• Single-molecule translation assays:

  • Combining with reconstituted translation systems to study ZNF598 function in vitro

  • Real-time monitoring of ubiquitination events during translation

  • Correlating with structural changes in ribosome conformation

• Quantitative proteomics integration:

  • SILAC or TMT labeling to quantify changes in the ZNF598 interactome

  • Identifying dynamic interaction partners during ribosome stalling

  • Mapping the complete ubiquitylation landscape dependent on ZNF598

• Genome-wide CRISPR screens:

  • Using ZNF598 antibodies to validate hits from genetic screens

  • Identifying synthetic lethal interactions with ZNF598

  • Uncovering new components of the RQC pathway

These methodological advances are enabling researchers to study ZNF598 function with unprecedented precision and in previously inaccessible contexts .

How do the detection properties of different commercial ZNF598 antibodies compare in various research applications?

A comparative analysis reveals significant differences between available antibodies:

When selecting between these options, researchers should consider:

• The specific experimental application

• Required species reactivity

• Need for consistency across experiments

• Availability of validation data relevant to their model system

How should researchers integrate ZNF598 antibody data with other approaches to comprehensively study ribosome-associated quality control?

A multi-modal approach provides the most comprehensive understanding:

• Genetic manipulation validation:

  • Compare antibody detection patterns with genetic ZNF598 knockdown/knockout

  • Use CRISPR-edited cell lines with epitope tags for validation

  • Consider rescue experiments with wild-type vs. mutant ZNF598

• Functional assay integration:

  • Combine antibody detection with reporter assays for ribosome stalling

  • Correlate ZNF598 localization with sites of stalled translation

  • Monitor both protein levels and activity (ubiquitination of substrates)

• Multi-omics correlation:

  • Link antibody-based detection with RNA-seq, Ribo-seq, and proteomics data

  • Identify transcripts most affected by ZNF598 manipulation

  • Correlate with global translation efficiency measurements

• Systems biology approaches:

  • Network analysis of ZNF598 interactors identified by antibody-based methods

  • Pathway enrichment analysis of differentially expressed genes/proteins

  • Mathematical modeling of RQC dynamics incorporating quantitative antibody data

• Evolutionary conservation studies:

  • Compare ZNF598 expression, localization, and function across species

  • Use antibodies against conserved epitopes for cross-species studies

  • Identify species-specific adaptations in RQC pathways

This integrated approach allows researchers to distinguish between correlation and causation in ZNF598 function studies and to place findings in broader biological context .

What are the emerging technological developments that will enhance ZNF598 antibody applications in the future?

Several technological advances promise to expand ZNF598 antibody applications:

• Engineered recombinant antibodies:

  • Single-domain antibodies (nanobodies) for improved access to conformational epitopes

  • Site-specific conjugation for precise labeling without affecting binding properties

  • Bispecific antibodies targeting ZNF598 and ribosomal proteins simultaneously

• Intracellular antibody fragments:

  • Cell-permeable antibody derivatives for live-cell imaging

  • Intrabodies for targeted manipulation of ZNF598 in specific cellular compartments

  • Proximity-dependent labeling combined with antibody recognition

• Single-molecule applications:

  • Direct visualization of ZNF598-ribosome interactions using antibody-based FRET pairs

  • Combined with in vitro translation systems for real-time monitoring

  • Correlation with structural changes during collision events

• Mass cytometry integration:

  • Metal-conjugated antibodies for high-dimensional analysis of RQC components

  • Single-cell proteomics to capture heterogeneity in RQC responses

  • Spatial mapping of ZNF598 distribution in tissues

• Computational prediction tools:

  • AI-assisted epitope prediction for optimal antibody design

  • Structure-based modeling of antibody-ZNF598 interactions

  • Integration with AlphaFold-predicted structural domains

These technological developments will enable more precise localization, quantification, and functional analysis of ZNF598 in diverse experimental contexts .

What key research questions about ZNF598 remain to be addressed, and how will antibody-based approaches contribute to answering them?

Critical unresolved questions include:

• Substrate specificity mechanisms:

  • How does ZNF598 distinguish collided from normal ribosomes?

  • Are there additional ribosomal protein targets beyond RPS10/RPS3/RPS20?

  • Antibodies against specific conformational states will help address these questions

• Regulatory mechanisms of ZNF598:

  • How is ZNF598 activity itself regulated (PTMs, localization, protein interactions)?

  • Does ZNF598 function change under different stress conditions?

  • Modification-specific antibodies will be crucial for studying these aspects

• Tissue-specific functions:

  • Does ZNF598 have specialized roles in different tissues/cell types?

  • Are there tissue-specific interaction partners?

  • IHC with validated antibodies across tissue panels will provide insights

• Disease associations:

  • How does ZNF598 function change in neurodegenerative disease or cancer?

  • Can ZNF598 activity be targeted therapeutically?

  • Antibodies will serve as both research tools and potential diagnostic markers

• Evolutionary adaptations:

  • How has ZNF598 function evolved across species?

  • Are there species-specific substrates or regulatory mechanisms?

  • Cross-reactive antibodies will enable comparative studies