rnasekb Antibody

What is rnasekb and what cellular functions does it participate in?

Rnasekb is the zebrafish (Danio rerio) ortholog of Ribonuclease K (RNASEK), an endoribonuclease that preferentially cleaves ApU and ApG phosphodiester bonds and hydrolyzes UpU bonds at a lower rate . RNASEK proteins play critical roles in:

• Regulating the activity of vacuolar (H+)-ATPase (V-ATPase), which is responsible for acidifying and maintaining pH of intracellular compartments

• Early stages of receptor-mediated endocytosis

• Facilitating both clathrin-mediated and clathrin-independent endocytic uptake of viruses, including dengue, West Nile, Sindbis, Rift Valley Fever, influenza, and human rhinoviruses

In zebrafish specifically, rnasekb has been identified in transcriptomic studies related to T-cell leukemia development and CNS infiltration, with a recorded fold change of 1.54 in certain experimental conditions .

What are the structural characteristics of rnasekb that researchers should consider when selecting antibodies?

When selecting antibodies against rnasekb, researchers should consider:

• Rnasekb is predicted to be an integral component of membrane

• The protein has RNA endonuclease activity with specific cleavage preferences

• Sequence homology between zebrafish rnasekb (UniProt: Q0P442) and other species should be considered when planning cross-reactivity studies

• Antibody epitope selection is critical - targeting conserved regions may increase cross-reactivity with other species, while targeting variable regions increases specificity

For optimal experimental design, consider the protein's membrane localization when planning cell permeabilization protocols for immunocytochemistry or flow cytometry applications.

What validation methods should be implemented before using a new rnasekb antibody lot?

A robust antibody validation strategy for rnasekb should include:

• Western blot analysis: Verify the antibody detects a band of the expected molecular weight in zebrafish samples

• Positive and negative controls: Include tissues/cells known to express and not express rnasekb

• Peptide competition assay: Confirm specificity by pre-incubating the antibody with the immunizing peptide

• Genetic validation: If possible, test antibody in rnasekb knockout/knockdown samples

• Cross-reactivity assessment: Test the antibody against related ribonucleases

As emphasized by the Antibody Characterization forum: "Antibody characterization is critical to enhance reproducibility and reliability of research findings... It has been estimated that ~50% of commercial antibodies fail to meet even basic standards for characterization" . Always document validation results thoroughly before proceeding with experiments.

What are the recommended protocols for Western blot optimization using rnasekb antibodies?

For optimal Western blot results with rnasekb antibodies:

• Sample preparation:

  • For membrane proteins like rnasekb, use detergent-based lysis buffers (e.g., RIPA with 1% NP-40 or Triton X-100)

  • Include protease inhibitors to prevent degradation

  • Heat samples at 70°C instead of 95°C to prevent membrane protein aggregation

• Gel selection and transfer:

  • Use 12-15% polyacrylamide gels for this smaller protein

  • PVDF membranes typically work better than nitrocellulose for hydrophobic membrane proteins

  • Include a low-methanol transfer buffer (5-10%)

• Antibody incubation:

  • Optimize primary antibody concentration (typically start with 1:500-1:2000)

  • Extended incubation at 4°C (overnight) may improve specific binding

  • Use 5% BSA instead of milk for blocking and antibody dilution

• Controls and validation:

  • Include positive control samples from tissue known to express rnasekb

  • Consider using rnaseka (paralog) expressing tissues as specificity controls

  • Validate antibody specificity using peptide competition assays

How can immunohistochemistry (IHC) protocols be optimized for rnasekb detection in zebrafish tissues?

For effective rnasekb detection in zebrafish tissues:

• Fixation optimization:

  • For zebrafish embryos/larvae: 4% PFA for 2-4 hours at room temperature

  • For adult tissues: 4% PFA overnight at 4°C

  • Avoid over-fixation which can mask epitopes

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • For membrane proteins like rnasekb, try proteolytic-induced epitope retrieval with proteinase K

• Permeabilization:

  • Critical for membrane proteins - use 0.5% Triton X-100 for 30 minutes

  • For whole-mount staining, extend permeabilization time to 1-2 hours

• Antibody dilution and incubation:

  • Test dilutions from 1:100 to 1:1000

  • Incubate 36-48 hours at 4°C for whole-mount preparations

  • Include 1% DMSO to improve tissue penetration

• Signal detection optimization:

  • For fluorescent detection, try TSA (tyramide signal amplification) for low abundance proteins

  • For chromogenic detection, increase DAB development time with close monitoring

What experimental designs are recommended for studying rnasekb's role in viral infection pathways?

Given rnasekb's involvement in viral entry pathways, consider these experimental approaches:

• Infection models:

  • Use zebrafish embryos infected with fluorescently labeled viruses

  • Compare viral entry in normal vs. rnasekb-depleted conditions

  • Track co-localization of rnasekb with viral particles using confocal microscopy

• Endocytosis assays:

  • Monitor uptake of labeled transferrin (clathrin-dependent) and cholera toxin B (clathrin-independent)

  • Compare endocytosis rates between control and rnasekb knockdown cells

  • Assess pH regulation in endosomal compartments using pH-sensitive dyes

• V-ATPase interaction studies:

  • Co-immunoprecipitation of rnasekb with V-ATPase components

  • Assess V-ATPase activity in rnasekb-depleted cells

  • Visualize co-localization using dual immunofluorescence

• Rescue experiments:

  • Reintroduce wild-type or mutant rnasekb to knockout models

  • Assess which domains are critical for viral entry functions

  • Compare human RNASEK and zebrafish rnasekb functional conservation

As shown in human RNASEK studies, "Required at an early stage of both clathrin-mediated and clathrin-independent endocytic uptake of a diverse set of viruses" , suggesting similar experimental approaches would be valuable for zebrafish rnasekb characterization.

How can CRISPR/Cas9 be combined with rnasekb antibodies for comprehensive functional studies?

An integrated approach using CRISPR/Cas9 and antibody detection provides robust functional insights:

• Generation of zebrafish rnasekb mutants:

  • Design sgRNAs targeting early exons of rnasekb

  • Create tissue-specific or inducible knockout models

  • Verify knockout efficiency using rnasekb antibodies

• Epitope tagging of endogenous rnasekb:

  • Use CRISPR/Cas9 to introduce FLAG, HA, or GFP tags

  • Compare tagged protein detection with native antibody detection

  • Validate antibody specificity against tagged vs. untagged controls

• Rescue experiments with structure-function analysis:

  • Reintroduce wild-type or domain-mutated rnasekb variants

  • Use antibodies to verify expression levels of rescue constructs

  • Correlate rescue protein levels with functional recovery

• Temporal analysis of rnasekb expression:

  • Combine with transgenic reporter lines for tissue-specific studies

  • Track expression during development or disease progression

  • Correlate with phenotypic outcomes in knockout models

These approaches allow for validation of antibody specificity while simultaneously generating valuable functional data on rnasekb biology.

What are the methodological considerations when using rnasekb antibodies in proximity ligation assays (PLA)?

When implementing PLA for rnasekb interaction studies:

• Antibody compatibility requirements:

  • Primary antibodies must be from different species (e.g., rabbit anti-rnasekb and mouse anti-V-ATPase)

  • Verify each antibody works independently in immunofluorescence first

  • Test antibody concentration ranges (typically 5-10× more dilute than for standard IF)

• Sample preparation optimization:

  • Critical fixation control: 4% PFA for 10-15 minutes

  • Mild permeabilization: 0.1-0.2% Triton X-100 to preserve membrane structure

  • Blocking with 5% BSA with 0.1% fish gelatin to reduce background

• Controls required for interpretation:

  • Single primary antibody controls (omit one antibody)

  • Non-interacting protein pair as negative control

  • Known interacting proteins as positive control

  • Include rnasekb-depleted samples as specificity control

• Quantification approaches:

  • Count PLA foci per cell using automated image analysis

  • Compare subcellular distribution of interaction signals

  • Correlate with functional assays measuring endocytosis or viral entry

This technique is particularly valuable for studying rnasekb's interactions with V-ATPase components or viral proteins in intact cells or tissues.

How can contradictory results between different rnasekb antibodies be reconciled and interpreted?

When facing contradictory results between different rnasekb antibodies:

• Epitope mapping analysis:

  • Determine the exact epitopes recognized by each antibody

  • Check for post-translational modifications that might affect epitope accessibility

  • Assess epitope conservation across species if using antibodies raised against different orthologs

• Validation with orthogonal approaches:

  • Confirm protein expression using mRNA detection methods (qRT-PCR, RNA-seq)

  • Use CRISPR/Cas9 to tag the endogenous protein for unambiguous detection

  • Employ mass spectrometry for protein identification

• Systematic comparison of antibody performance:

  • Test all antibodies under identical conditions

  • Document specific protocol differences that affect performance

  • Create a comparison table of results across multiple detection methods

• Collaborative validation:

  • As emphasized in search result : "Two independent experimental laboratories" can provide stronger evidence

  • Implement a standardized evaluation protocol similar to the approach described for in-silico generated antibodies

Example comparison table for contradictory antibody results - hypothetical data for illustration purposes

What quantitative approaches should be used to analyze rnasekb expression in Western blots and immunostaining?

For robust quantitative analysis of rnasekb expression:

• Western blot quantification:

  • Use appropriate loading controls (β-actin for whole cell, Na+/K+-ATPase for membrane fractions)

  • Implement a standard curve of recombinant protein for absolute quantification

  • Employ technical triplicates and biological replicates (n≥3)

  • Statistical analysis: normalized band intensities using ANOVA with post-hoc tests

• Immunofluorescence quantification:

  • Capture images with identical acquisition parameters

  • Measure mean fluorescence intensity within defined cellular compartments

  • Count positive cells as percentage of total population

  • Use Z-stack acquisitions for volume-based quantification

• Flow cytometry analysis:

  • Gate on live, single cells before measuring rnasekb signal

  • Include fluorescence-minus-one (FMO) controls

  • Report median fluorescence intensity rather than mean

  • Use histogram overlays to visualize population shifts

• Tissue expression mapping:

  • Implement whole-slide scanning for tissue-wide expression patterns

  • Use machine learning approaches for unbiased quantification

  • Correlate expression with tissue/cell type markers

For all methods, follow the standards for antibody validation described in search result : "The ability to detect, quantify, enrich, localize, and/or perturb the function of a target protein – even when present in a complex protein mixture... is key to many biomedical research studies."

How should researchers interpret differences in rnasekb staining patterns between subcellular compartments?

When analyzing differential subcellular localization of rnasekb:

• Membrane vs. cytoplasmic staining:

  • Membrane localization correlates with active endocytic functions

  • Cytoplasmic signal may represent newly synthesized protein or internalized membrane

  • Quantify ratio of membrane:cytoplasmic signal for functional state assessment

• Endosomal localization patterns:

  • Early endosome localization (co-stain with EEA1): active endocytosis

  • Late endosome/lysosome localization (co-stain with LAMP1): protein degradation

  • Recycling endosome localization (co-stain with Rab11): protein recycling

• Nuclear signal interpretation:

  • Verify with additional antibodies and fractionation studies

  • Investigate potential nuclear functions with ChIP or RNA-binding assays

  • Consider antibody cross-reactivity with nuclear ribonucleases

• Dynamic relocalization during stimulation:

  • Monitor time-course after viral exposure or pH changes

  • Quantify kinetics of redistribution between compartments

  • Correlate with functional assays (endocytosis rates, viral entry)

As indicated in search result , RNASEK "regulates the activity of vacuolar (H+)-ATPase (V-ATPase) which is responsible for acidifying and maintaining the pH of intracellular compartments," suggesting that subcellular localization is functionally significant.

What controls are essential when publishing research using rnasekb antibodies?

When publishing research using rnasekb antibodies, include these essential controls:

• Antibody validation controls:

  • Genetic controls: rnasekb knockout/knockdown samples

  • Peptide competition/blocking experiments

  • Multiple antibodies against different epitopes

  • Recombinant protein expression as positive control

• Technical controls:

  • Secondary-only controls for background assessment

  • Isotype controls for non-specific binding

  • Concentration-matched controls for all primary antibodies

  • Validated housekeeping proteins as loading controls

• Biological controls:

  • Developmental time-course (temporal control)

  • Tissue panel (spatial control)

  • Treatment response (functional control)

  • Cross-species comparison for evolutionary conservation

• Reporting standards:

  • Complete antibody information (vendor, catalog #, lot #, RRID)

  • Detailed methods including concentrations and incubation times

  • Raw, unprocessed blot/image data in supplementary materials

  • Quantification method with statistical analysis

As emphasized in search result : "It has been estimated that ~50% of commercial antibodies fail to meet even basic standards for characterization, and this problem is thought to result in financial losses of $0.4–1.8 billion per year in the United States alone." Proper controls and validation are therefore essential for research integrity and reproducibility.