wu:fa18f11 Antibody

What is the wu:fa18f11 protein and what biological systems express it?

The wu:fa18f11 protein (UniProt Number: Q4KMC9) is an uncharacterized protein from Danio rerio (zebrafish) . While its complete characterization is still ongoing in the research community, it represents one of many zebrafish proteins being studied as part of developmental biology research. The protein is primarily expressed in zebrafish, though homologs may exist in related species. Understanding this protein contributes to our knowledge of vertebrate development, as zebrafish serve as an important model organism for studying gene function due to their transparent embryos and rapid development.

What applications is the wu:fa18f11 antibody validated for in research settings?

The wu:fa18f11 antibody has been validated for several research applications, primarily:

For researchers planning to use this antibody in other applications such as immunohistochemistry (IHC), immunocytochemistry (ICC), or flow cytometry, preliminary validation experiments are strongly recommended as these applications are not explicitly listed in the current validation data.

What are the optimal storage conditions and stability characteristics of the wu:fa18f11 antibody?

The wu:fa18f11 antibody should be stored at either -20°C or -80°C for long-term preservation of activity . The antibody formulation contains 0.03% Proclin 300 as a preservative and 50% glycerol as a stabilizer , which helps maintain antibody integrity during freeze-thaw cycles.

For researchers working with this antibody:

• Aliquot upon first thaw to minimize freeze-thaw cycles

• When working with the antibody, keep it on ice

• Return to -20°C or -80°C promptly after use

• Monitor for signs of degradation such as precipitation or loss of activity in control experiments

How can researchers validate the specificity of the wu:fa18f11 antibody in their experimental systems?

Validating antibody specificity is essential for experimental rigor. For the wu:fa18f11 antibody, which targets a zebrafish protein, consider these validation approaches:

• Positive and negative controls: Use recombinant Danio rerio wu:fa18f11 protein as a positive control . The antibody product comes with 200μg of recombinant immunogen protein that can serve as this positive control . Additionally, the supplied 1ml pre-immune serum can function as a negative control .

• Knock-down/Knock-out validation: If possible, use morpholino technology or CRISPR-Cas9 to knock down or knock out the wu:fa18f11 gene in zebrafish, then confirm reduced or absent antibody signal.

• Signal specificity testing: Compare patterns of reactivity with those reported in literature or predicted based on mRNA expression data from zebrafish genomic databases.

• Cross-reactivity assessment: Test the antibody on tissues where the target protein is not expected to be expressed.

This multi-faceted approach to validation follows principles similar to those used in validating broadly neutralizing antibodies in other research areas, where extensive characterization is required to confirm specificity and function .

What are the considerations for cross-species reactivity of the wu:fa18f11 antibody?

The wu:fa18f11 antibody is specifically raised against Danio rerio (zebrafish) protein . When considering cross-species applications:

• Sequence homology analysis: Before testing in other species, compare the sequence homology of the immunogen used to raise the antibody with potential homologs in target species. Higher sequence conservation suggests higher likelihood of cross-reactivity.

• Epitope mapping: If the specific epitope recognized by the antibody is known, analyze its conservation across species.

• Empirical testing protocol:

  • Begin with Western blot as it typically requires less optimization than IHC

  • Use positive controls from zebrafish alongside samples from the target species

  • Consider using higher antibody concentrations initially (2-5x recommended dilution)

  • Include pre-adsorption controls with the immunizing peptide

The antibody was generated using recombinant protein as the immunogen , which may recognize conformational epitopes, potentially limiting cross-reactivity to species with highly conserved three-dimensional protein structures.

How can researchers optimize wu:fa18f11 antibody performance for challenging experimental conditions?

Optimization strategies for the wu:fa18f11 antibody, particularly in challenging experimental conditions:

• Sample preparation optimization:

  • For fixed tissues: Test different fixation protocols (4% PFA vs. methanol)

  • For protein extraction: Compare different lysis buffers with varying detergent strengths

  • For membrane proteins: Consider specialized extraction buffers containing appropriate detergents

• Antibody incubation conditions:

  • Temperature variations: 4°C overnight vs. room temperature for 1-2 hours

  • Buffer optimization: Try different blocking agents (BSA, milk, serum)

  • Signal enhancement: Consider using amplification systems like tyramide signal amplification for low-abundance targets

• Data from optimization experiments:

Similar optimization approaches have proven successful in studies of broadly neutralizing antibodies where detection of specific epitopes required careful experimental design .

What is the recommended protocol for using wu:fa18f11 antibody in Western blot applications?

For optimal Western blot results with the wu:fa18f11 antibody:

• Sample preparation:

  • Prepare zebrafish tissue lysates in RIPA buffer containing protease inhibitors

  • Heat samples at 95°C for 5 minutes in Laemmli buffer containing DTT

  • Load 20-40 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels based on the expected molecular weight

  • Transfer to PVDF membrane at 100V for 1 hour in standard transfer buffer

• Antibody incubation:

  • Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

  • Dilute primary antibody according to manufacturer's recommendations

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3x10 minutes with TBST

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature

  • Wash 3x10 minutes with TBST

  • Develop using enhanced chemiluminescence

• Controls to include:

  • Positive control: Recombinant wu:fa18f11 protein (provided with antibody)

  • Negative control: Pre-immune serum (provided with antibody)

  • Loading control: Anti-GAPDH or anti-β-actin antibody

This approach parallels methodologies used in studies characterizing novel antibodies against target proteins, where rigorous controls are essential for result interpretation .

How should researchers design experiments to study wu:fa18f11 expression during zebrafish development?

Developmental expression studies require careful experimental design:

• Temporal expression analysis:

  • Collect embryos at key developmental stages (e.g., 4-cell, shield, 24 hpf, 48 hpf, 72 hpf)

  • Process each stage for both protein (Western blot, IHC) and mRNA (qPCR, in situ hybridization) analysis

  • Compare protein detection by wu:fa18f11 antibody with mRNA expression patterns

• Spatial expression assessment:

  • Perform whole-mount immunostaining using wu:fa18f11 antibody

  • Use confocal microscopy for precise localization

  • Consider double-staining with tissue-specific markers to identify expressing cell types

• Functional studies:

  • Design morpholino knockdown or CRISPR knockout experiments

  • Assess phenotypic consequences

  • Verify knockdown/knockout efficiency using the wu:fa18f11 antibody

• Expression analysis data organization:

This developmental approach mirrors methodologies used in functional genomics analyses of antibody expression patterns in other systems .

What are effective troubleshooting strategies for non-specific binding when using wu:fa18f11 antibody?

When encountering non-specific binding with wu:fa18f11 antibody:

• Blocking optimization:

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

  • Increase blocking time from 1 hour to 2-3 hours

  • Consider adding 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform a titration series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

  • Balance specific signal intensity against background

  • Consider longer incubation with more dilute antibody

• Washing stringency:

  • Increase number of washes (from 3x to 5-6x)

  • Extend wash duration (from 5 min to 10-15 min)

  • Add higher salt concentration to wash buffer (increase NaCl from 150mM to 250-500mM)

• Pre-adsorption controls:

  • Pre-incubate antibody with the immunizing peptide/protein before application

  • This should eliminate specific binding while leaving non-specific binding intact

  • Use the included recombinant immunogen protein for this purpose

These troubleshooting approaches align with methodologies used in antibody characterization studies where specificity determination is crucial .

How can researchers quantitatively assess wu:fa18f11 protein levels in different experimental conditions?

For quantitative assessment of wu:fa18f11 protein levels:

• Western blot quantification:

  • Use a standard curve of recombinant wu:fa18f11 protein (supplied with antibody)

  • Employ densitometry software for band intensity quantification

  • Normalize to loading controls (GAPDH, β-actin)

  • Analyze biological replicates (minimum n=3) for statistical validity

• ELISA-based quantification:

  • Develop a sandwich ELISA using wu:fa18f11 antibody

  • Generate a standard curve using the supplied recombinant protein

  • Ensure sample dilutions fall within the linear range of the standard curve

  • Include technical triplicates for each biological sample

• Flow cytometry for single-cell analysis (if applicable):

  • Optimize fixation and permeabilization for intracellular detection

  • Use fluorophore-conjugated secondary antibodies

  • Include fluorescence-minus-one (FMO) controls

  • Analyze median fluorescence intensity (MFI) rather than percentage positive

• Comparative quantification example:

Similar quantitative approaches have been employed in studies characterizing antibodies against target proteins where precise measurement of binding affinities and expression levels was critical .

How might wu:fa18f11 antibody be used in comparative studies between zebrafish and other model organisms?

The wu:fa18f11 antibody presents opportunities for evolutionary and comparative studies:

• Evolutionary conservation assessment:

  • Test cross-reactivity with homologous proteins in other fish species (medaka, stickleback)

  • Examine functional conservation by comparing expression patterns across species

  • Correlate protein localization with gene expression data from related species

• Methodological approach for cross-species studies:

  • Identify putative homologs through bioinformatic analysis

  • Align protein sequences to identify conserved domains

  • Design experiments testing antibody reactivity in multiple species under identical conditions

  • Validate findings with species-specific genetic approaches (knockout/knockdown)

• Data integration strategy:

  • Create comparative expression maps across developmental stages

  • Correlate findings with transcriptomic data from multiple species

  • Employ phylogenetic analyses to interpret differences in antibody reactivity

This comparative approach reflects methodologies used in evolutionary studies of conserved proteins, where antibodies targeting specific epitopes can reveal functional conservation across species .

What considerations are important when designing immunoprecipitation experiments with wu:fa18f11 antibody?

While immunoprecipitation (IP) is not explicitly listed among the validated applications, researchers might explore this approach with careful optimization:

• Pre-experiment considerations:

  • Evaluate if the polyclonal nature of the antibody is advantageous for capturing native protein

  • Consider coupling the antibody to protein A/G beads for clean IP

  • Plan for appropriate controls (IgG control, input sample)

• Optimization parameters:

  • Lysis buffer composition (detergent type and concentration)

  • Antibody amount (typically 1-5 μg per reaction)

  • Incubation conditions (overnight at 4°C vs. shorter times)

  • Washing stringency balance (maintain specific interactions while removing non-specific binding)

• Validation of IP results:

  • Confirm specific pull-down via Western blot

  • Consider mass spectrometry to identify co-immunoprecipitated proteins

  • Validate key interactions with reciprocal IP experiments

• IP-optimization experimental design:

Such IP approaches have been crucial in studies identifying protein-protein interactions in complex biological systems .

How can researchers integrate wu:fa18f11 antibody-based protein detection with transcriptomic data analysis?

Integrating protein and transcriptomic data provides a more comprehensive understanding of gene function:

• Multi-omics experimental design:

  • Collect matched samples for both protein analysis (using wu:fa18f11 antibody) and RNA-seq

  • Process samples in parallel to minimize technical variation

  • Include appropriate time points to capture dynamic regulation

• Correlation analysis methodology:

  • Quantify protein levels via Western blot or ELISA using wu:fa18f11 antibody

  • Normalize RNA-seq data and calculate gene expression values (TPM/FPKM)

  • Perform correlation analysis between protein and mRNA levels

  • Identify instances of concordant and discordant regulation

• Biological interpretation framework:

  • For concordant changes: likely transcriptional regulation

  • For protein changes without mRNA changes: post-transcriptional regulation

  • For mRNA changes without protein changes: possible translational control

• Integration analysis example:

This integrative approach mirrors functional genomics analyses used in studies of gene expression regulation, where correlations between transcript and protein levels provide insights into regulatory mechanisms .