zgc:101851 Antibody

What is zgc:101851 and why is it important in zebrafish research?

zgc:101851 refers to a protein coding gene in Danio rerio (zebrafish) that has been identified through genomic sequencing projects. It is part of the zebrafish genome collection (zgc) and is cataloged with UniProt accession number Q66IB8 . Zebrafish serve as excellent model organisms for studying vertebrate development and disease due to their transparent embryos, rapid development, and genetic similarity to humans.

The functional characterization of zgc:101851 remains an active area of investigation. Antibodies against this protein enable researchers to track its expression patterns during development and in various tissues, contributing to our understanding of its biological role. As with many zebrafish proteins, studying zgc:101851 may provide insights into conserved biological processes relevant to human health and disease.

What applications are validated for the zgc:101851 antibody?

According to product specifications, the zgc:101851 antibody has been tested and validated for several research applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of the zgc:101851 protein in solution .

• Western Blotting (WB): For detection of zgc:101851 in protein extracts from zebrafish tissues .

The antibody is typically provided as a polyclonal antibody raised in rabbit against recombinant Danio rerio zgc:101851 protein . When designing experiments with this antibody, researchers should carefully consider sample preparation methods, blocking solutions, and detection systems compatible with the antibody's properties. As with any research antibody, validation for your specific experimental conditions is essential, as performance can vary based on tissue fixation methods, protein denaturation conditions, and other experimental variables .

What are the optimal storage and handling conditions for zgc:101851 antibody?

Based on manufacturer specifications, the optimal storage conditions for zgc:101851 antibody are:

• Temperature: Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles, as this can degrade antibody quality and affect performance

• Storage buffer composition: The antibody is typically provided in liquid form with 50% Glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

For daily use, prepare small aliquots and store at -20°C to minimize freeze-thaw cycles. When working with the antibody, keep it on ice or at 4°C. Long-term stability can be affected by improper storage, so it's advisable to retest antibody performance if the storage time exceeds the manufacturer's recommendations.

Maintaining proper documentation of storage conditions, including receipt date, lot number, and aliquoting information, is essential for troubleshooting and reproducibility .

How should I validate the specificity of zgc:101851 antibody?

Validating antibody specificity is crucial for generating reliable research data. For zgc:101851 antibody, implement these methodological approaches:

• Genetic Validation:

  • Use CRISPR/Cas9 to generate zgc:101851 knockout zebrafish

  • Compare antibody staining between wild-type and knockout samples

  • Absence of signal in knockout samples strongly indicates specificity

• Molecular Validation:

  • Perform Western blot to confirm a single band of expected molecular weight

  • Pre-absorb the antibody with recombinant zgc:101851 protein before staining

  • Signal should be significantly reduced or eliminated after pre-absorption

• Orthogonal Validation:

  • Compare protein detection with an alternative method (e.g., mass spectrometry)

  • Use a second antibody targeting a different epitope of zgc:101851

  • Concordant results strengthen confidence in antibody specificity

• Expression Pattern Validation:

  • Compare antibody staining patterns with known mRNA expression data

  • Discrepancies may indicate non-specific binding

These validation steps align with the guidelines established by the International Working Group for Antibody Validation and should be thoroughly documented in your research methods .

What controls should be included when using zgc:101851 antibody?

When conducting experiments with zgc:101851 antibody, include these controls to ensure reliable and interpretable results:

• Positive Controls:

  • Samples known to express zgc:101851 (specific zebrafish tissues or developmental stages)

  • Recombinant zgc:101851 protein (if available)

• Negative Controls:

  • No primary antibody control (secondary antibody only)

  • Isotype control (using a non-specific IgG from the same species)

  • Ideally, tissues from zgc:101851 knockout zebrafish

• Blocking Peptide Control:

  • Pre-incubate the antibody with the immunizing peptide

  • Should abolish specific staining if the antibody is binding specifically

• Cross-Application Validation:

  • If using the antibody for a new application, validate results using an orthogonal method

  • For example, confirm immunohistochemistry findings with Western blot

According to best practices in antibody validation, these controls are essential for establishing confidence in experimental results and ensuring reproducibility .

How can I optimize Western blot protocols specifically for zgc:101851 antibody?

Optimizing Western blot protocols for zgc:101851 antibody requires systematic adjustment of several parameters:

For troubleshooting:

• High background: Increase blocking time, dilute antibodies further, or add 0.1-0.5% Tween-20 to washing buffer

• No signal: Check protein transfer efficiency, reduce antibody dilution, or increase exposure time

• Multiple bands: Increase stringency with higher NaCl concentration in wash buffer, or pre-absorb with similar proteins

Always normalize signal to appropriate loading controls (β-actin, GAPDH, or total protein staining) and document all optimization steps in publications.

What are the known cross-reactivity issues with zgc:101851 antibody?

Understanding potential cross-reactivity is essential for accurate data interpretation:

• Predicted Cross-Reactivity:

  • The antibody is raised against recombinant Danio rerio zgc:101851 protein, so it may cross-react with structurally similar proteins within zebrafish

  • Sequence homology analysis can predict potential cross-reactivity with related proteins

  • The antibody is not expected to cross-react with mammalian proteins, but this should be empirically verified if working across species

• Empirical Verification Approaches:

  • Western blot analysis might reveal bands at unexpected molecular weights, indicating potential cross-reactivity

  • Pre-adsorption experiments with related proteins can help identify specific cross-reactivity

  • Mass spectrometry identification of immunoprecipitated proteins can uncover unexpected targets

• Minimizing Cross-Reactivity Effects:

  • Increase washing stringency in immunoassays

  • Optimize antibody concentration to minimize non-specific binding

  • Pre-clear lysates with Protein A/G beads before immunoprecipitation

  • Validate results with orthogonal methods that don't rely on antibody specificity

It's critical to document any observed cross-reactivity in your methods section and include full-length blots in publications, not just the region of interest .

How do I troubleshoot non-specific binding in immunohistochemistry with zgc:101851 antibody?

Non-specific binding in immunohistochemistry can compromise data interpretation. Use this methodological approach when troubleshooting:

• Optimize Blocking Conditions:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Triton X-100 to permeabilize tissues effectively

• Antibody Dilution Series:

  • Perform a titration from 1:100 to 1:2000 to identify optimal concentration

  • Higher dilutions often reduce background but may also reduce specific signal

  • Find the balance that maximizes signal-to-noise ratio

• Secondary Antibody Considerations:

  • Ensure secondary antibody is raised against the host species of zgc:101851 antibody (typically rabbit)

  • Pre-adsorb secondary antibody against zebrafish tissues

  • Use highly cross-adsorbed secondary antibodies to minimize cross-reactivity

• Technical Modifications:

  • Optimize fixation protocol (duration, fixative composition)

  • Increase washing duration and frequency (5 × 10 minutes with agitation)

  • Consider antigen retrieval methods if signal is weak but specific

• Control Experiments:

  • Include secondary-only controls to assess non-specific binding

  • Pre-absorb primary antibody with immunizing peptide

  • Test the antibody on tissues known not to express zgc:101851

Document all optimization parameters and take images with identical settings for valid comparisons across conditions .

How can I design experiments to study zgc:101851 function in zebrafish development?

To investigate the developmental role of zgc:101851, consider these methodological approaches:

• Expression Pattern Analysis:

  • Perform whole-mount immunohistochemistry with zgc:101851 antibody at different developmental stages

  • Combine with in situ hybridization for zgc:101851 mRNA to compare protein and mRNA localization

  • Create a detailed spatiotemporal expression map from fertilization through organogenesis

• Loss-of-Function Studies:

  • Generate CRISPR/Cas9 knockout zebrafish lines targeting zgc:101851

  • Design morpholinos for transient knockdown

  • Document phenotypes at key developmental stages

  • Perform rescue experiments by injecting mRNA to confirm specificity

• Gain-of-Function Approaches:

  • Create mRNA for microinjection at the one-cell stage

  • Generate transgenic lines with tissue-specific overexpression

  • Document developmental abnormalities or altered development

• Molecular Pathway Analysis:

  • Perform pull-down assays with zgc:101851 antibody to identify interaction partners

  • Conduct RNA-seq on knockout vs. wild-type embryos at key developmental stages

  • Use pathway analysis software to identify affected signaling networks

• Experimental Design Considerations:

  • Include appropriate numbers of embryos (n≥30 per condition)

  • Blind scorers to genotype during phenotypic analysis

  • Use multiple founders to control for insertion site effects in transgenics

  • Document genetic background of zebrafish lines used

Each approach should include appropriate controls and statistical analysis, with methods detailed thoroughly in publications.

How can I use zgc:101851 antibody to study protein interactions in zebrafish models?

Studying protein interactions using zgc:101851 antibody requires careful experimental design:

• Co-Immunoprecipitation (Co-IP):

  • Prepare zebrafish tissue lysates under non-denaturing conditions

  • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

  • Incubate cleared lysates with zgc:101851 antibody overnight at 4°C

  • Capture immune complexes with Protein A/G beads

  • Elute and analyze by mass spectrometry or Western blot for interacting partners

  • Include IgG control and reverse Co-IP for validation

• Proximity Ligation Assay (PLA):

  • Fix zebrafish tissues using paraformaldehyde

  • Incubate with zgc:101851 antibody and antibody against potential interacting protein

  • Use species-specific PLA probes followed by rolling circle amplification

  • Visualize fluorescent spots indicating <40nm proximity of proteins

  • Quantify interaction signals across different tissues or conditions

• Pull-down Assays:

  • Express recombinant zgc:101851 with affinity tag

  • Incubate with zebrafish lysates

  • Capture complexes and identify binding partners by mass spectrometry

  • Validate interactions using zgc:101851 antibody in Western blot

• Data Analysis and Validation:

  • Create interaction networks using bioinformatics tools

  • Validate key interactions using multiple techniques

  • Assess functional relevance through genetic studies

  • Compare interaction data with known pathways in zebrafish

These approaches provide complementary information about zgc:101851's interactome in zebrafish models, contributing to understanding its biological function.

What information should I include about zgc:101851 antibody in my publications?

To ensure reproducibility and transparency in research involving zgc:101851 antibody, include the following information in your publications:

• Complete Antibody Information:

  • Antibody name and target (zgc:101851)

  • Vendor/source (e.g., Cusabio, Nordic Biosite)

  • Catalog number (e.g., CSB-PA721079XA01DIL)

  • Lot number (critical for batch-to-batch variation assessment)

  • Host species (typically rabbit)

  • Clonality (polyclonal/monoclonal)

  • Immunogen information (e.g., recombinant Danio rerio zgc:101851)

• Methodological Details:

  • Antibody dilution used for each application

  • Incubation conditions (time, temperature)

  • Blocking reagents and conditions

  • Detection methods and systems

  • Complete protocol or reference to detailed protocol

• Validation Data:

  • Include validation experiments performed

  • Present controls used (positive, negative, blocking peptide)

  • Show full-length blots, not just cropped images of bands of interest

  • Include validation across applications if the antibody was used in multiple assays

• Quantification Methods:

  • Describe image acquisition parameters

  • Detail quantification methods and software used

  • Specify statistical analyses performed

  • Report sample sizes and biological replicates

Following these guidelines aligns with international initiatives to improve antibody reporting standards and enhances the reproducibility of your research .

How do batch-to-batch variations affect zgc:101851 antibody experiments?

Batch-to-batch variation is a significant concern with research antibodies, particularly polyclonal antibodies like those typically used against zgc:101851:

• Sources of Variation:

  • Polyclonal antibodies represent a heterogeneous mixture of antibodies targeting different epitopes

  • Different animal immunizations produce different antibody repertoires

  • Manufacturing processes may have variable consistency

  • Storage conditions and antibody age can affect performance

• Impact on Experiments:

  • Signal intensity may vary between batches

  • Background levels may differ

  • Epitope recognition patterns might change

  • Cross-reactivity profiles could be altered

• Mitigation Strategies:

  • Purchase sufficient quantity of a single lot for complete projects

  • Validate each new antibody lot before use in critical experiments

  • Include lot number in all experimental records and publications

  • Maintain reference samples to compare antibody performance between lots

• Documentation Practices:

  • Create a validation protocol specific to your experimental system

  • Document performance metrics for each lot

  • Store images of standard samples labeled with different lots

  • Share batch validation data when publishing or presenting results

Recognizing that batch-to-batch variation is inherent in antibody production, especially for polyclonal antibodies, proper validation and documentation are essential to maintain experimental consistency and reproducibility .