zgc:65873 Antibody

What are the basic characteristics of commercially available zgc:65873 antibodies?

The zgc:65873 antibody is available as a rabbit polyclonal antibody purified by antigen affinity chromatography . Key characteristics include:

• Type: Polyclonal

• Host: Rabbit

• Isotype: IgG

• Applications: ELISA and Western Blot (WB)

• Immunogen: Recombinant Danio rerio (Zebrafish) zgc:65873 protein

• Conjugation status: Unconjugated

• Recommended storage conditions: -20°C or -80°C

How should zgc:65873 antibody be validated before experimental use?

Proper validation is essential for experimental reliability. A methodological approach should include:

• Positive and negative controls: Use the provided antigens (200μg) as a positive control and pre-immune serum (1ml) as a negative control .

• Specificity testing: Run parallel assays with closely related proteins to ensure the antibody does not cross-react.

• Concentration optimization: Perform titration experiments to determine optimal antibody concentration.

• Western blot validation: Confirm that the antibody detects a band of expected molecular weight.

• ELISA validation: Similar to approaches used for other antibodies such as those detailed in ZIKV studies, where coated plates are blocked, and antibodies are diluted serially in blocking buffer before detection with secondary antibodies .

How can zgc:65873 antibody be integrated into comprehensive developmental studies in zebrafish?

For advanced developmental research:

• Timeline expression analysis: Use the antibody to track zgc:65873 expression at different developmental stages (embryonic to adult).

• Tissue-specific localization: Combine with whole-mount immunohistochemistry to map expression patterns in specific tissues and organs.

• Comparative expression studies: Similar to approaches used for other genes in zebrafish studies, researchers can examine expression in various organs including "the brain, eyes, olfactory bulb, branchial arches, heart, liver, intestine or gut, pronephos, somites, lateral lines, pectoral fins, caudal fin fold, trunk and tail blood vessels, dermal epithelium, and yolk syncytial layer" .

• Response to environmental factors: Examine how zgc:65873 expression changes under different environmental conditions, similar to methodologies used in toxicological studies with zebrafish .

What methodological approaches can be used to study potential binding partners of zgc:65873?

Researchers can employ several advanced techniques:

• Co-immunoprecipitation (Co-IP): Use purified zgc:65873 antibody to pull down the protein and its binding partners from zebrafish tissue lysates.

• Proximity-based labeling: Adapt methods like BioID or APEX to identify proteins in close proximity to zgc:65873.

• Yeast display technologies: Similar to those used in antibody studies, these can be adapted to identify binding partners, as demonstrated in antigen specificity studies .

• Cross-linking followed by immunoprecipitation: Use chemical cross-linkers to stabilize transient protein-protein interactions before immunoprecipitation with zgc:65873 antibody.

• NGS-based functional characterization: Adapt sequencing approaches to study protein interactions, similar to methods used for antibody repertoire analysis .

How can functional genomics approaches be combined with zgc:65873 antibody studies?

Integration of functional genomics approaches may include:

• Correlation of protein expression with transcriptomic data: Compare zgc:65873 protein levels (measured by antibody-based techniques) with mRNA expression patterns from microarray or RNA-seq data.

• CRISPR-Cas9 genetic modifications: Generate zgc:65873 knockouts or mutations, then use the antibody to confirm protein depletion or modification.

• Morpholino studies: Design morpholinos to transiently knock down zgc:65873 and use the antibody to confirm knockdown efficiency.

• Transgenic reporter lines: Create reporter lines for zgc:65873 and validate expression patterns using the antibody.

• Global gene expression analysis: Similar to microarray approaches used in zebrafish studies that identified "464 MeHg-up-regulated and 379 MeHg-down-regulated genes" , researchers can examine how zgc:65873 expression correlates with other genes under various conditions.

What are the optimal protocols for using zgc:65873 antibody in Western blotting?

Based on general antibody methodologies and the specific properties of this antibody:

What are the key methodological considerations for zgc:65873 antibody in ELISA applications?

For optimal ELISA performance:

• Coating: Coat ELISA plates with 100 ng/well of purified recombinant zgc:65873 protein or zebrafish tissue lysate.

• Blocking: Block with SuperBlock or equivalent for 2 hours at room temperature.

• Antibody dilution: Create a serial dilution series of zgc:65873 antibody to determine optimal concentration.

• Detection: Use anti-rabbit HRP-conjugated secondary antibody (approximately 1:20,000 dilution).

• Development: Add appropriate substrate (e.g., Super AquaBlue ELISA substrate) and incubate for 10 minutes before stopping the reaction with 1M sulfuric acid.

• Absorbance reading: Measure at 405 nm .

How can researchers optimize immunohistochemistry protocols with zgc:65873 antibody?

For effective immunohistochemistry:

• Tissue preparation: Fix zebrafish embryos or tissues in 4% paraformaldehyde.

• Permeabilization: Treat with 0.1-0.5% Triton X-100 to enhance antibody penetration.

• Antigen retrieval: Test different methods (heat-induced, enzymatic) to determine optimal retrieval.

• Blocking: Use 5-10% normal goat serum to reduce non-specific binding.

• Primary antibody incubation: Incubate with zgc:65873 antibody at optimized dilution (start with 1:100-1:500) overnight at 4°C.

• Secondary detection: Use fluorescently-labeled or enzyme-conjugated anti-rabbit secondary antibodies.

• Counterstaining: Include DAPI for nuclear visualization.

• Controls: Always include appropriate positive and negative controls as provided with the antibody package .

How can researchers address weak or absent signals when using zgc:65873 antibody?

When signal issues occur:

• Verify antibody viability: Ensure proper storage conditions (-20°C or -80°C) and check expiration date.

• Increase antibody concentration: Try serial dilutions to find optimal concentration.

• Extend incubation time: Increase primary antibody incubation from overnight to 24-48 hours at 4°C.

• Enhance antigen retrieval: Test alternative antigen retrieval methods.

• Reduce washing stringency: Use milder washing conditions to prevent antibody removal.

• Check sample preparation: Ensure protein denaturation is optimal for exposing the epitope.

• Test positive control: Use the provided antigen positive control (200μg) to verify antibody functionality .

What approaches can address non-specific binding or high background with zgc:65873 antibody?

To improve specificity:

• Optimize blocking: Increase blocking time or concentration, or test alternative blocking agents.

• Increase washing steps: Add more washing steps with longer durations.

• Dilute antibody: Use higher dilutions of primary and secondary antibodies.

• Pre-absorb antibody: Incubate with non-specific proteins before application to samples.

• Use provided negative control: Incorporate the pre-immune serum provided as a negative control .

• Filter lysates: Remove debris that may cause non-specific binding.

• Reduce detection time: Shorten substrate incubation time for enzymatic detection methods.

How can researchers validate questionable zgc:65873 antibody results?

Validation approaches include:

• Alternative detection methods: If Western blot shows unexpected results, verify with ELISA or vice versa.

• Genetic validation: Use morpholinos or CRISPR to knockdown/knockout zgc:65873 and confirm antibody specificity.

• Mass spectrometry: Identify proteins recognized by the antibody using immunoprecipitation followed by mass spectrometry.

• Alternative antibodies: If available, test different antibodies against zgc:65873 or epitope-tagged versions.

• Cross-species reactivity: Although the antibody is specific to zebrafish , checking reactivity in closely related species may provide insight into specificity.

• RNA-protein correlation: Compare protein detection with mRNA expression data from techniques like in situ hybridization, similar to approaches used in zebrafish studies showing "68% positive correlation with the microarray result" .

How can zgc:65873 antibody be utilized in toxicological studies with zebrafish?

For toxicological applications:

• Dose-response experiments: Examine zgc:65873 expression changes at various toxicant concentrations.

• Time-course studies: Track protein expression changes over different exposure durations.

• Tissue-specific responses: Analyze expression changes in specific tissues using immunohistochemistry.

• Comparative toxicology: Study zgc:65873 responses across different toxicants.

• Molecular pathway analysis: Combine with other markers to map toxicant-induced pathway changes.

This approach parallels zebrafish toxicological studies where researchers have identified genes with "sensitive expression changes" that "may serve as marker genes for detecting the presence of [toxicants] in the environment" .

What approaches enable quantitative analysis of zgc:65873 expression levels?

For quantitative expression analysis:

• Quantitative Western blot: Use increasing amounts of recombinant zgc:65873 protein to create a standard curve.

• Quantitative ELISA: Develop a sandwich ELISA using the zgc:65873 antibody and a secondary detection antibody.

• Image analysis software: Quantify immunohistochemistry signals using appropriate software.

• Flow cytometry: Adapt the antibody for intracellular staining in dissociated zebrafish cells.

• Automated plate reader analysis: For high-throughput screening of zgc:65873 expression across multiple conditions.

These methods allow researchers to quantify expression similar to approaches used for real-time PCR quantification of gene expression in zebrafish studies .

How can zgc:65873 antibody be adapted for high-throughput screening applications?

For high-throughput applications:

• Automated ELISA: Develop robotics-assisted ELISA protocols for zgc:65873 detection across large sample sets.

• Tissue microarrays: Create zebrafish tissue microarrays for rapid immunohistochemical screening.

• Automated Western blot systems: Utilize automated protein separation and detection systems.

• Multiplex assays: Combine zgc:65873 antibody with other antibodies for simultaneous detection of multiple proteins.

• High-content imaging: Integrate with automated microscopy systems for cellular localization studies.

These approaches parallel high-throughput antibody screening technologies used in other studies to "determine comprehensive functional molecular and genetic profiles" .