zgc:110130 Antibody

What is zgc:110130 and what is its function in zebrafish?

zgc:110130 is a protein-coding gene found in zebrafish (Danio rerio). According to genomic databases, it is involved in nucleobase-containing compound transmembrane transport . Expression analyses show that zgc:110130 is expressed in various developmental stages of zebrafish embryos, including at 28hpf and 40hpf in trunk and whole embryo samples . The protein is part of a larger network of genes expressed during zebrafish development.

To investigate its function, researchers typically employ techniques including:

• In situ hybridization to visualize expression patterns

• Knockout models to observe phenotypic effects

• Immunohistochemistry using antibodies to detect protein localization

• Expression correlation studies with other genes

How are antibodies against zebrafish proteins generally developed?

Antibodies against zebrafish proteins are typically developed through several approaches:

• Recombinant protein expression: The zebrafish target protein (or immunogenic fragments) is expressed in bacterial, insect, or mammalian expression systems, purified, and used as an immunogen.

• Synthetic peptide approach: Short peptide sequences unique to the zebrafish protein are synthesized and conjugated to carrier proteins like KLH (Keyhole Limpet Hemocyanin) before immunization.

• Genetic immunization: DNA constructs encoding the target zebrafish protein are directly administered, resulting in in vivo expression of the antigen.

For zgc:110130 specifically, researchers would likely:

• Analyze the protein sequence for immunogenic epitopes

• Design expression constructs or synthetic peptides

• Immunize host animals (typically rabbits for polyclonal or mice for monoclonal antibodies)

• Screen and validate the resulting antibodies using techniques such as Western blotting, immunohistochemistry, and knockout controls .

What are the typical applications for zgc:110130 antibodies in zebrafish research?

Antibodies against zgc:110130 would typically be used in:

• Developmental biology studies: Tracking protein expression throughout zebrafish development

• Gene function studies: Correlating protein presence with phenotypic outcomes

• Subcellular localization: Determining where the protein functions within cells

• Protein interaction studies: Identifying binding partners through co-immunoprecipitation

• Cell-type specific expression: Determining which cell types express the protein

Based on gene expression data, zgc:110130 shows positive correlation with neuronal genes like sncb, gng3, and stxbp1a, suggesting potential roles in neuronal tissues . Antibodies would be valuable for confirming these expression patterns at the protein level.

What validation approaches are recommended for zebrafish antibodies?

Validation of zebrafish antibodies, including those targeting zgc:110130, should follow multiple orthogonal approaches:

• Genetic knockout validation: Testing antibody in wild-type and knockout samples is the gold standard. Recent large-scale antibody validation studies show this approach identifies many non-specific commercial antibodies .

• Expression pattern validation: Comparing antibody staining with known mRNA expression patterns.

• Multiple antibody verification: Using multiple antibodies targeting different epitopes of zgc:110130.

• Epitope competition: Using purified peptides to block antibody binding.

• Cross-reactivity testing: Ensuring the antibody doesn't detect closely related zebrafish proteins.

For zebrafish research specifically, validation typically includes immunohistochemistry on tissue sections or whole-mount preparations with appropriate controls .

How can I assess the specificity of a commercially available zgc:110130 antibody?

To rigorously assess the specificity of a zgc:110130 antibody:

• Western blot analysis: Perform Western blots on zebrafish tissue lysates to verify binding to a protein of expected molecular weight. The predicted molecular weight should be determined from the amino acid sequence of zgc:110130.

• Knockout/knockdown controls: If available, use CRISPR/Cas9 knockout or morpholino knockdown zebrafish as negative controls. The complete absence of signal in knockouts indicates specificity .

• Overexpression verification: Express tagged zgc:110130 in cells and verify antibody co-localization with the tag.

• Cross-species reactivity: Test reactivity with human or mouse homologs if cross-reactivity is claimed.

• Peptide blocking: Pre-incubate antibody with the immunizing peptide to verify signal reduction.

Recent large-scale validation studies demonstrate that approximately 50% of commercial antibodies fail in one or more applications, highlighting the importance of thorough validation .

What alternative approaches exist when antibodies against zgc:110130 show cross-reactivity?

When dealing with cross-reactive antibodies against zgc:110130:

• Epitope binning and refinement: Employ Epitope Binning-seq technology to characterize antibody binding sites and develop more specific antibodies. This approach enables simultaneous evaluation of large numbers of genetically encoded antibodies targeting different epitopes .

• Tagged protein expression: Generate transgenic zebrafish expressing tagged versions of zgc:110130 (e.g., GFP or FLAG tags).

• Mass spectrometry validation: Combine immunoprecipitation with mass spectrometry to identify all proteins captured by the antibody .

• Orthogonal detection methods: Use RNA-based detection methods (in situ hybridization, RNAscope) to complement antibody staining.

• Recombinant antibody engineering: Develop highly specific recombinant antibodies, which generally show better performance than traditional monoclonal or polyclonal antibodies .

How can I optimize immunohistochemistry protocols for zgc:110130 detection in zebrafish retina?

Optimizing immunohistochemistry for zgc:110130 in zebrafish retina requires:

• Fixation optimization:

  • For adult retina: 4% paraformaldehyde for 2-4 hours at 4°C

  • For embryonic tissue: 2% paraformaldehyde for 1-2 hours

  • Test alternative fixatives if standard methods fail (e.g., Dent's fixative)

• Antigen retrieval methods:

  • Heat-mediated antigen retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

  • Enzymatic retrieval using proteinase K (especially for whole-mount samples)

• Blocking optimization:

  • Use 10% goat serum with 1% BSA and 0.3% Triton X-100

  • Add 0.1% fish gelatin to reduce background in retinal tissues

• Antibody incubation:

  • Test different concentrations (typically 2-5 μg/ml for commercial antibodies)

  • Extend incubation time to 48-72 hours at 4°C for whole-mount samples

  • For sections, overnight incubation is typically sufficient

• Signal amplification:

  • Consider using tyramide signal amplification for low-abundance targets

  • HRP-conjugated secondary antibodies with DAB can provide sensitive detection

Recent studies on zebrafish retinal regeneration demonstrate successful antibody staining protocols for detecting proteins in rod photoreceptors .

What strategies can improve antibody-based detection of zgc:110130 in Western blots?

For optimal Western blot detection of zgc:110130:

• Sample preparation optimization:

  • Test multiple lysis buffers (RIPA, NP-40, or specialized extraction buffers)

  • Include protease inhibitors to prevent degradation

  • Consider subcellular fractionation if the protein localizes to specific compartments

• Gel percentage selection:

  • Based on predicted molecular weight, choose appropriate acrylamide percentage

  • Consider gradient gels (4-20%) for better resolution

• Transfer optimization:

  • For smaller proteins: use PVDF membranes and shorter transfer times

  • For larger proteins: use nitrocellulose membranes and longer/lower voltage transfers

• Blocking optimization:

  • Test different blocking agents (5% milk, 3-5% BSA, commercial blockers)

  • Optimize blocking time (1-3 hours at room temperature or overnight at 4°C)

• Antibody dilution optimization:

  • Test a range of primary antibody dilutions (typically 1:500 to 1:5000)

  • Incubate membranes overnight at 4°C for optimal binding

• Detection system selection:

  • Enhanced chemiluminescence (ECL) for standard detection

  • Fluorescent secondary antibodies for multiplex detection and quantification

Recent research on zebrafish proteins demonstrates successful Western blot protocols with antibody dilutions around 1:5000 and detection using peroxidase-conjugated secondary antibodies .

How can I evaluate protein-protein interactions of zgc:110130 using antibodies?

To investigate protein-protein interactions involving zgc:110130:

• Co-immunoprecipitation (Co-IP):

  • Use anti-zgc:110130 antibodies to pull down the protein complex

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • Include appropriate controls: IgG control, lysate from knockout samples

• Proximity ligation assay (PLA):

  • Detect protein interactions in situ with single-molecule sensitivity

  • Requires antibodies from different species against zgc:110130 and potential interacting partners

  • Results in fluorescent dots where proteins interact within 40 nm

• Immunofluorescence co-localization:

  • Perform dual immunofluorescence with antibodies against zgc:110130 and potential partners

  • Analyze co-localization using confocal microscopy and quantitative co-localization analysis

• FRET/FLIM analysis:

  • Use fluorophore-conjugated antibodies for Förster Resonance Energy Transfer

  • Requires careful controls and specialized equipment

• Crosslinking-assisted IP:

  • Chemically crosslink protein complexes before immunoprecipitation

  • Helps capture transient or weak interactions

Based on gene correlation data, potential interaction partners for zgc:110130 might include proteins encoded by genes showing positive correlation, such as sncb (r=0.242), gng3 (r=0.240), and ywhag2 (r=0.235) .

What are common pitfalls when using zgc:110130 antibodies in zebrafish research?

Common pitfalls when using antibodies against zebrafish proteins like zgc:110130 include:

• Inadequate validation:

  • Failure to validate antibody specificity in zebrafish tissues

  • Relying solely on vendor data without independent validation

  • Research indicates that more than 50% of commercial antibodies fail in one or more applications

• Fixation-dependent epitope masking:

  • Different fixation methods can dramatically affect epitope accessibility

  • Test multiple fixation protocols if initial staining fails

• Cross-reactivity with related proteins:

  • Zebrafish genome duplication has resulted in paralogs that may cross-react

  • Validate specificity against related zebrafish proteins

• Developmental stage-specific expression:

  • zgc:110130 expression varies across developmental stages

  • Ensure sampling at appropriate developmental timepoints

• Tissue-specific optimization requirements:

  • Protocols may need significant modification for different zebrafish tissues

  • Permeabilization requirements differ between embryonic and adult tissues

• Background in zebrafish yolk:

  • High autofluorescence and non-specific binding in yolk

  • Use appropriate blocking reagents and consider deyolking procedures

How can I discriminate between different isoforms of zgc:110130 using antibodies?

To discriminate between potential isoforms of zgc:110130:

• Isoform-specific antibody design:

  • Design antibodies against unique regions of specific isoforms

  • Target exon junctions present only in certain splice variants

  • According to ZFIN, zgc:110130 has at least two mRNA variants (zgc:110130-201 and zgc:110130-202)

• Western blot optimization:

  • Use high-resolution SDS-PAGE to separate closely sized isoforms

  • Consider using Phos-tag gels to separate phosphorylated isoforms

  • Optimize running conditions for maximum separation of similar molecular weight proteins

• Isoform-specific knockdown controls:

  • Design morpholinos or CRISPR guides specific to individual isoforms

  • Use these knockdowns to validate antibody specificity

• Combined immunoprecipitation and mass spectrometry:

  • Immunoprecipitate with antibodies recognizing all isoforms

  • Identify specific isoforms through peptide mass fingerprinting

  • This approach can identify post-translational modifications distinguishing isoforms

• Comparison with isoform-specific RNA detection:

  • Correlate antibody staining with isoform-specific in situ hybridization

  • Use RT-PCR with isoform-specific primers alongside protein detection

What considerations are important when designing new antibodies against zgc:110130?

When designing new antibodies against zgc:110130, consider:

• Epitope selection criteria:

  • Target regions with high predicted antigenicity and surface exposure

  • Avoid transmembrane domains and regions with post-translational modifications

  • Target unique regions not present in related zebrafish proteins

  • Consider evolutionary conservation if cross-species reactivity is desired

• Production platform selection:

  • Recombinant monoclonal antibodies generally perform better than hybridoma-derived monoclonals or polyclonals

  • Consider phage display libraries for difficult targets

  • For conformational epitopes, use full-length protein immunization

• Host species considerations:

  • Choose host species based on planned applications

  • Rabbit antibodies often provide higher affinity but may have more background in zebrafish

  • Consider chicken antibodies for reduced background in zebrafish tissues

• Validation strategy planning:

  • Design knockout controls before antibody generation

  • Plan for epitope binning to identify antibodies targeting different regions

  • Novel Epitope Binning-seq technology enables evaluation of large numbers of antibodies simultaneously

• Application-specific design:

  • For immunohistochemistry: target epitopes resistant to fixation

  • For Western blotting: target linear epitopes

  • For immunoprecipitation: target surface-exposed regions

How can advanced computational approaches improve zgc:110130 antibody specificity prediction?

Advanced computational approaches for improving antibody specificity include:

• Epitope prediction algorithms:

  • Utilize BepiPred, DiscoTope, and other tools to identify optimal epitopes

  • Incorporate protein structure predictions using AlphaFold to identify surface-exposed regions

  • For zgc:110130, this would involve analyzing its predicted structure to identify optimal antibody targets

• Cross-reactivity prediction:

  • Perform BLAST searches against the entire zebrafish proteome to identify similar sequences

  • Use epitope mapping tools to avoid regions with homology to other proteins

  • Analyze the correlation between zgc:110130 and other genes to identify potential biological relationships

• Active learning strategies for antibody development:

  • Implement machine learning approaches for antibody-antigen binding prediction

  • Active learning strategies can reduce the number of required experimental validations by up to 35%

  • Library-on-library approaches allow identification of specific interacting pairs

• Molecular dynamics simulations:

  • Predict epitope flexibility and accessibility in solution

  • Model antibody-antigen interactions to optimize binding

• Integration with experimental validation pipelines:

  • Combine in silico predictions with high-throughput experimental validation

  • Incorporate feedback from validation experiments to improve future predictions

  • Recent validation pipelines have demonstrated the efficiency of standardized characterization approaches

How should I interpret conflicting results between antibody-based detection and mRNA expression data for zgc:110130?

When facing discrepancies between antibody detection and mRNA expression:

• Consider post-transcriptional regulation:

  • mRNA levels may not correlate directly with protein levels due to translation efficiency

  • Analyze the correlation patterns of zgc:110130 with potential regulatory genes

• Evaluate temporal dynamics:

  • Protein often appears later than mRNA due to translation time

  • In zebrafish development, examine slightly later timepoints for protein compared to peak mRNA expression

• Assess spatial differences:

  • Proteins may be transported away from the site of synthesis

  • Compare subcellular localization in antibody staining with in situ hybridization patterns

• Examine technical factors:

  • Antibody sensitivity may differ from mRNA detection methods

  • Fixation might affect epitope accessibility differently across tissues

• Consider protein stability:

  • Long-lived proteins may persist after mRNA levels decrease

  • Short-lived proteins may be difficult to detect despite high mRNA levels

• Design validation experiments:

  • Use reporter constructs to track protein production and localization

  • Perform pulse-chase experiments to assess protein stability

  • Consider relevant biological contexts based on gene correlation data showing zgc:110130 may function in neuronal contexts

What experimental design is recommended for studying zgc:110130 function using antibody approaches?

A comprehensive experimental design would include:

• Expression profiling:

  • Temporal expression: Analyze zgc:110130 expression across developmental stages

  • Spatial expression: Map expression patterns across tissues and cell types

  • Correlate with expression of functionally related genes identified through correlation analysis

• Loss-of-function studies:

  • Generate CRISPR/Cas9 knockouts of zgc:110130

  • Use morpholino knockdowns for early developmental stages

  • Analyze phenotypes with antibodies against pathway components

• Protein interaction studies:

  • Perform co-immunoprecipitation with anti-zgc:110130 antibodies

  • Identify interaction partners through mass spectrometry

  • Validate interactions with co-localization studies

• Subcellular localization:

  • Use immunofluorescence to determine precise subcellular localization

  • Perform fractionation followed by Western blotting

  • Correlate localization with potential functions

• Functional rescue experiments:

  • Rescue knockout/knockdown phenotypes with wild-type or mutant constructs

  • Use antibodies to verify expression of rescue constructs

  • Correlate rescue efficiency with protein levels

• Pathway analysis:

  • Use antibodies against pathway components to assess effects of zgc:110130 manipulation

  • Perform phospho-specific antibody analysis to assess signaling changes

Consider targeting genes with high correlation to zgc:110130, such as sncb (r=0.242) and gng3 (r=0.240), in parallel experiments to understand potential functional relationships .

How can I integrate antibody-based approaches with other methodologies for comprehensive characterization of zgc:110130?

For comprehensive characterization, integrate multiple approaches:

• Multi-omics integration:

  • Combine antibody-based proteomics with transcriptomics and genomics

  • Correlate protein expression with RNA-seq data

  • Use CRISPR screens to identify functional relationships

• Live imaging with antibody fragments:

  • Develop Fab fragments or nanobodies for live imaging

  • Track protein dynamics in real-time

  • Combine with fluorescent reporters for multi-channel imaging

• Functional assays with protein modulation:

  • Use degron-based approaches for acute protein depletion

  • Correlate phenotypes with protein levels detected by antibodies

  • Employ optogenetic tools to control protein function

• Structural biology integration:

  • Use antibodies as crystallization chaperones

  • Perform hydrogen-deuterium exchange mass spectrometry with antibody binding

  • Correlate structural information with functional domains

• Single-cell analysis:

  • Combine antibody staining with single-cell RNA-seq

  • Analyze protein heterogeneity across cell populations

  • Create high-resolution expression maps during development

• Disease models:

  • Study zgc:110130 in zebrafish disease models

  • Use antibodies to assess expression changes in pathological states

  • Correlate with human disease markers