zgc:112255 Antibody

What is zgc:112255 and why is it of interest to researchers?

Zgc:112255 is a protein-coding gene in zebrafish (Danio rerio) located on chromosome 11. It is orthologous to human C1orf50 (chromosome 1 open reading frame 50) and encodes a 189-amino acid protein (UniProtKB:Q502G5) . The protein contains a domain of unknown function DUF2452 (InterPro ID: IPR019534) . Its conservation across species suggests functional importance, making it an interesting target for developmental biology and comparative genomics research. The gene was previously named si:dkey-208k8.3 before receiving its current designation .

What applications are zgc:112255 antibodies suitable for?

Zgc:112255 antibodies have been validated for several common laboratory techniques. Available research-grade antibodies are suitable for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) . These applications allow researchers to detect and quantify zgc:112255 protein in tissue samples, cell lysates, and other biological specimens. While immunohistochemistry and immunofluorescence applications are not explicitly mentioned in the search results, these techniques may be possible with appropriate validation by individual researchers.

What expression patterns does zgc:112255 exhibit in zebrafish?

According to expression data from Thisse et al. (2004), zgc:112255 shows specific expression patterns during zebrafish development . The gene exhibits correlated expression with several nucleolar and ribosomal processing genes, including npm1a (r=0.126), snu13b (r=0.119), nop58 (r=0.117), and dkc1 (r=0.114) . Conversely, it shows negative correlation with neuronal markers like elavl3 (r=-0.058), gpm6aa (r=-0.056), and neuronal-specific genes . This expression profile suggests potential roles in ribosome biogenesis or nucleolar function during development.

How can I determine the specificity of a zgc:112255 antibody?

To determine antibody specificity, implement a multi-step validation approach. First, perform Western blot analysis using zebrafish tissue lysates to confirm the antibody detects a protein of the expected molecular weight (~21 kDa for zgc:112255). Second, include appropriate negative controls such as pre-immune serum or isotype controls. Third, use positive controls where the protein is known to be expressed based on Thisse expression data . For definitive validation, perform knockdown experiments using morpholinos or CRISPR-Cas9 targeting zgc:112255, which should result in reduced or absent signal. Additionally, overexpression of tagged zgc:112255 can serve as a positive control to further verify antibody specificity.

What is the optimal sample preparation method for detecting zgc:112255 in zebrafish tissues?

For optimal detection of zgc:112255 in zebrafish tissues, implement a sample preparation protocol that preserves protein integrity while maximizing extraction efficiency. For Western blotting, homogenize tissues in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) supplemented with protease inhibitors. For embryonic samples, deyolk embryos before lysis to reduce background. When preparing samples for immunohistochemistry, fix tissues in 4% paraformaldehyde for 24 hours at 4°C, followed by paraffin embedding or cryopreservation. For whole-mount applications, permeabilize fixed embryos with 0.5% Triton X-100 to facilitate antibody penetration. When designing experiments, consider the correlated expression patterns with nucleolar proteins like npm1a and nop58 , which might inform the developmental stages and tissues most likely to yield robust zgc:112255 detection.

How should I optimize Western blot conditions for zgc:112255 detection?

For optimal Western blot detection of zgc:112255, consider the protein's biochemical properties derived from its sequence. Load 20-50 μg of total protein per lane on a 12-15% SDS-PAGE gel to ensure good resolution of the ~21 kDa target protein. After transfer to a PVDF or nitrocellulose membrane (0.2 μm pore size recommended for small proteins), block with 5% non-fat dry milk in TBST for 1 hour at room temperature. Dilute the primary zgc:112255 antibody at 1:500 to 1:2000 in blocking solution and incubate overnight at 4°C . After washing, apply an appropriate HRP-conjugated secondary antibody at 1:5000 dilution. For enhanced sensitivity, consider using signal amplification systems or fluorescent secondary antibodies with digital imaging. If detection remains challenging, optimize by adjusting antibody concentration, incubation time, or using alternative blocking agents like BSA or commercial blockers that might reduce background while preserving specific binding.

What controls should be included when using zgc:112255 antibodies in immunostaining experiments?

A comprehensive control strategy for immunostaining with zgc:112255 antibodies should include multiple types of controls. Primary controls should include: (1) Omission of primary antibody to assess secondary antibody non-specific binding; (2) Isotype controls using non-specific IgG at the same concentration as the zgc:112255 antibody; (3) Absorption controls where the antibody is pre-incubated with excess recombinant zgc:112255 protein before staining. Biological controls should include: (1) Positive control tissues based on known expression patterns from Thisse et al. data ; (2) Negative control tissues where the protein is not expected to be expressed; (3) Morpholino or CRISPR knockdown samples where zgc:112255 expression is reduced. Additional verification can be achieved by comparing staining patterns with in situ hybridization results for zgc:112255 mRNA, which should show similar tissue distribution patterns to protein detection by immunostaining.

How can I use zgc:112255 antibodies to investigate protein-protein interactions?

To investigate protein-protein interactions involving zgc:112255, implement co-immunoprecipitation (Co-IP) protocols optimized for this protein. First, prepare zebrafish tissue or cell lysates under non-denaturing conditions using a gentle lysis buffer (150 mM NaCl, 1% NP-40 or Triton X-100, 50 mM Tris pH 7.4) supplemented with protease inhibitors. Pre-clear lysates with Protein A/G beads before incubating with zgc:112255 antibody (5 μg per mg of protein lysate) overnight at 4°C. Capture antibody-protein complexes using Protein A/G beads, wash extensively, and elute for analysis by Western blot. Based on correlation data showing associations with nucleolar proteins like npm1a (r=0.126), nop58 (r=0.117), and dkc1 (r=0.114) , these proteins are good candidates to test for interactions. Additionally, consider proximity ligation assays (PLA) for in situ detection of protein interactions in fixed tissues, which can provide spatial information about where within cells these interactions occur.

What strategies can address potential cross-reactivity with similar proteins when using zgc:112255 antibodies?

Addressing cross-reactivity concerns with zgc:112255 antibodies requires a multi-faceted approach focusing on epitope selection and validation. Since zgc:112255 contains a domain of unknown function (DUF2452) , antibodies targeting this region might cross-react with other proteins containing similar domains. To minimize cross-reactivity: (1) Select antibodies raised against unique peptide sequences within zgc:112255 that have low homology to other zebrafish proteins; (2) Perform comprehensive bioinformatic analysis to identify potential cross-reactive proteins based on sequence similarity; (3) Validate specificity using knockout/knockdown models where zgc:112255 is absent; (4) Conduct Western blots across multiple tissues to identify any unexpected bands that might indicate cross-reactivity; (5) If available, test the antibody on a protein array containing various zebrafish proteins to directly assess cross-reactivity profiles. For critical applications, consider using two antibodies targeting different epitopes of zgc:112255 and confirming signal co-localization.

How can I use zgc:112255 antibodies to investigate its potential role in developmental processes?

To investigate zgc:112255's developmental role using antibodies, implement a comprehensive temporal and spatial analysis strategy. First, perform whole-mount immunostaining across key developmental stages (4-cell, shield, 5-somite, 24 hpf, 48 hpf, 72 hpf, and 5 dpf) to establish the protein's expression timeline. Process confocal z-stacks to generate 3D reconstructions of expression patterns. Based on correlation data showing positive associations with ribosomal processing genes (npm1a, nop58, dkc1) and negative correlations with neuronal markers (elavl3, gpm6aa) , focus on examining nucleolar localization and potential exclusion from neural tissues. Complement antibody studies with functional approaches by performing morpholino knockdown or CRISPR/Cas9 knockout of zgc:112255, then use the antibody to confirm protein depletion and analyze resulting phenotypes. Co-immunostaining with markers for proliferation, differentiation, and apoptosis can reveal potential functional roles. Additionally, consider proximity ligation assays with correlated proteins to identify developmental stage-specific interactions.

What considerations are important when developing zgc:112255 antibodies for multiplexed imaging applications?

Developing zgc:112255 antibodies for multiplexed imaging requires careful consideration of several technical factors. First, select the appropriate antibody isotype and species origin to ensure compatibility with other antibodies in your multiplex panel; rabbit polyclonal or rat monoclonal antibodies often work well in combination with mouse antibodies against other targets. Second, choose optimal fluorophore conjugation strategies—direct conjugation with small fluorophores (Alexa Fluor, DyLight, or Cy dyes) minimizes steric hindrance while providing spectral separation. Third, validate antibody performance after conjugation, as fluorophore attachment can sometimes affect binding properties. Fourth, implement proper controls for each multiplex combination to identify any unexpected cross-reactivity or signal interference. Based on the correlation data , consider designing multiplex panels that include nucleolar markers (npm1a, nop58) for co-localization studies, as these showed positive correlation with zgc:112255 expression (r=0.126 and r=0.117, respectively). For highly complex multiplexing, consider sequential imaging approaches using antibody stripping and reprobing protocols or spectral imaging with linear unmixing to separate overlapping fluorophore signals.

What are common challenges with zgc:112255 antibody immunoprecipitation and how can they be addressed?

Common challenges in zgc:112255 immunoprecipitation experiments include low protein yield, non-specific binding, and antibody performance issues. To address these: (1) Optimize lysis conditions—use NP-40 or Triton X-100 based buffers (avoiding harsh detergents like SDS) with salt concentrations between 100-150 mM to preserve protein-protein interactions while minimizing background; (2) Increase starting material, especially considering that zgc:112255 may have stage-specific or tissue-specific expression patterns ; (3) Pre-clear lysates thoroughly with Protein A/G beads before adding the antibody to reduce non-specific binding; (4) Cross-link the antibody to beads using dimethyl pimelimidate (DMP) to prevent antibody co-elution; (5) Consider a sequential immunoprecipitation approach where the supernatant from a first IP is subjected to a second round of immunoprecipitation to increase yield. If the antibody performs poorly in IP applications, try alternative epitope antibodies or consider using a tagged version of zgc:112255 with well-validated tag antibodies as an alternative approach.

How can I improve signal-to-noise ratio when using zgc:112255 antibodies in zebrafish tissue immunohistochemistry?

Improving signal-to-noise ratio in zgc:112255 immunohistochemistry requires optimization of multiple parameters. First, optimize tissue fixation—test 2%, 4%, and PFA fixation durations (4h, 8h, overnight) to identify conditions that best preserve epitopes while maintaining tissue morphology. Second, implement effective antigen retrieval—for paraffin sections, try citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) at 95-100°C for 10-20 minutes; for frozen sections, test 0.1% Triton X-100, 0.5% SDS, or 100% methanol for permeabilization. Third, optimize blocking—test 5-10% normal serum from the secondary antibody species supplemented with 1-3% BSA and 0.1-0.3% Triton X-100. Fourth, adjust antibody concentration—perform titration experiments with primary antibody dilutions ranging from 1:100 to 1:1000 to identify the optimal concentration that maximizes specific signal while minimizing background. Fifth, extend washing steps—implement 5-6 washes of 10 minutes each with gentle agitation. Finally, consider signal amplification systems like tyramide signal amplification (TSA) if the target protein is expressed at low levels, which might be the case for zgc:112255 in certain tissues or developmental stages.

What strategies can help detect low-abundance zgc:112255 protein in specific cell types?

Detecting low-abundance zgc:112255 protein in specific cell types requires specialized approaches focused on sensitivity and specificity. First, implement signal amplification techniques such as tyramide signal amplification (TSA), which can increase sensitivity 10-100 fold over conventional detection methods. Second, use highly sensitive detection systems like enhanced chemiluminescence (ECL) Plus or SuperSignal West Femto for Western blots. Third, consider sample enrichment strategies—for specific cell populations, use fluorescence-activated cell sorting (FACS) or laser capture microdissection prior to protein extraction to concentrate the cell type of interest. Fourth, implement proximity ligation assay (PLA) technology, which can detect single protein molecules through rolling circle amplification. Fifth, reduce background through careful optimization of blocking conditions—test different blockers including normal serum, BSA, casein, or commercial blockers to identify optimal formulations. Based on correlation data showing associations with nucleolar proteins , focus initial detection efforts on cell types with high nucleolar activity. For developmental studies, prioritize stages with known zgc:112255 expression based on Thisse et al. data .

What are the best practices for storing and handling zgc:112255 antibodies to maintain activity?

To maintain optimal zgc:112255 antibody activity, implement comprehensive storage and handling practices tailored to antibody formulation. For long-term storage, aliquot antibodies in small volumes (10-20 μL) immediately upon receipt to avoid repeated freeze-thaw cycles, which can lead to aggregation and loss of activity. Store at -20°C or -80°C depending on manufacturer recommendations, with -80°C preferred for long-term storage beyond 6 months. For working solutions, store at 4°C with preservatives (0.02-0.05% sodium azide) for up to 2 weeks. Avoid exposing antibodies to extreme pH conditions, high temperatures, or excessive agitation that can cause denaturation. When thawing frozen aliquots, allow them to thaw completely at 4°C rather than at room temperature or with artificial heating. Before each use, centrifuge antibody vials briefly to collect solution at the bottom and ensure homogeneity. For diluted working solutions, use high-quality, sterile-filtered buffer systems appropriate for the application. Consider implementing the QTY code modification approach for improved stability if custom antibody production is an option , as this has been shown to reduce aggregation propensity while maintaining binding affinity.

How can zgc:112255 antibodies be used to investigate potential roles in disease models?

Utilizing zgc:112255 antibodies in disease models requires a translational approach linking zebrafish research to human disease. First, establish the expression profile of zgc:112255 in relevant zebrafish disease models, particularly those affecting tissues where correlation data indicates expression . Second, perform co-immunoprecipitation studies to identify interaction partners that might link zgc:112255 to disease-relevant pathways. Third, use zgc:112255 antibodies to assess protein localization changes in disease states compared to healthy controls. Since zgc:112255 is orthologous to human C1orf50 , focus on disease models relevant to known or suspected C1orf50 functions in humans. For inflammatory disease models, investigate potential involvement in immune complex formation similar to rheumatoid factor responses, where T-cell dependencies and Fc gamma receptor interactions play crucial roles . In particular, assess whether zgc:112255 is involved in complement-independent, Fc gamma receptor-dependent processes by comparing protein expression and localization in wild-type versus Fc gamma receptor-deficient models. Quantitative immunohistochemistry and automated image analysis can provide robust metrics for comparing zgc:112255 expression across different disease models and treatment conditions.

How might zgc:112255 antibodies be adapted for in vivo imaging applications?

Adapting zgc:112255 antibodies for in vivo imaging applications requires modifications to enhance tissue penetration, stability, and signal generation while minimizing background. First, consider antibody fragment generation—convert full IgG antibodies to Fab or F(ab')2 fragments through enzymatic digestion, or develop single-chain variable fragments (scFv) to improve tissue penetration. Second, implement site-specific conjugation of near-infrared fluorophores (e.g., Alexa Fluor 750, IRDye 800CW) that provide deeper tissue penetration and reduced autofluorescence. Third, explore antibody-nanoparticle conjugates such as quantum dots or upconversion nanoparticles that offer enhanced photostability and signal strength. For vascular delivery in zebrafish larvae, microinject labeled antibodies into the circulation at 48-72 hpf when the blood-brain barrier is not fully formed. For direct tissue imaging, develop transgenic zebrafish lines expressing fluorescent protein-tagged zgc:112255 as an alternative approach that avoids antibody delivery challenges. When designing experiments, consider the correlated expression with nucleolar proteins , which might provide guidance on optimal developmental stages and tissues for in vivo imaging.

What approaches can combine zgc:112255 antibodies with advanced molecular techniques for comprehensive functional studies?

Integrating zgc:112255 antibodies with advanced molecular techniques creates powerful platforms for functional characterization. First, combine ChIP-seq with zgc:112255 immunoprecipitation to identify potential DNA binding sites if the protein has uncharacterized nuclear functions, which is suggested by its correlation with nucleolar proteins . Second, implement APEX2 proximity labeling by creating fusion proteins with zgc:112255 and the engineered ascorbate peroxidase APEX2, then use zgc:112255 antibodies to confirm proper localization of the fusion protein before proximity labeling. Third, develop CRISPR-based transcriptional modulation systems (CRISPRa/CRISPRi) targeting zgc:112255, then use antibodies to quantify resulting protein level changes and correlate with phenotypic outcomes. Fourth, employ high-content screening approaches where zgc:112255 antibody staining serves as a readout for genetic or chemical screens aimed at identifying regulators of its expression, localization, or modification. Finally, implement spatial transcriptomics followed by protein validation with zgc:112255 antibodies to create multi-omics datasets that comprehensively map both transcript and protein distributions across tissues. These integrated approaches can reveal functions beyond what either antibody-based or molecular techniques could discover independently.

How can correlation analysis data inform experimental design using zgc:112255 antibodies?

The correlation analysis data showing relationships between zgc:112255 and other genes provides valuable guidance for experimental design. Strong positive correlations with nucleolar and ribosomal processing genes (npm1a, r=0.126; snu13b, r=0.119; nop58, r=0.117) suggest potential roles in RNA processing or ribosome biogenesis. Design co-localization experiments using zgc:112255 antibodies alongside markers for these positively correlated proteins to determine if they share subcellular localization. The negative correlations with neuronal markers (elavl3, r=-0.058; gpm6aa, r=-0.056) suggest potential exclusion from neuronal lineages; test this hypothesis with dual immunostaining for zgc:112255 and neuronal markers across developmental stages.

This correlation-guided approach ensures that experimental designs target the most biologically relevant contexts for zgc:112255 function, increasing the likelihood of meaningful discoveries while optimizing resource utilization.