si:dkey-29f10.1 Antibody

What is si:dkey-29f10.1 and why is it studied in zebrafish research?

Si:dkey-29f10.1 is a protein-coding gene in zebrafish (Danio rerio) that has been cataloged in genomic databases but remains functionally uncharacterized. Researchers study this protein primarily through antibody-based detection methods to understand its expression patterns during development and in adult tissues. Unlike many well-characterized zebrafish genes, si:dkey-29f10.1 belongs to a class of genes identified through genomic sequencing that lack clear orthology with mammalian genes, making it particularly interesting for understanding zebrafish-specific biological processes .

The commercially available antibodies against this target (such as CSB-PA744228XA01DIL) provide researchers with tools to study this protein's expression and function through techniques like Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA) .

What detection methods are validated for si:dkey-29f10.1 antibody in zebrafish studies?

According to available research and product documentation, the si:dkey-29f10.1 antibody has been validated for:

• Western blotting (WB): Effective for determining protein expression levels and molecular weight verification

• ELISA: Suitable for quantitative measurement of si:dkey-29f10.1 protein in solution

Unlike some other zebrafish antibodies that have been validated for immunohistochemistry (IHC) or immunofluorescence (IF), current documentation doesn't specifically claim validation for these applications with si:dkey-29f10.1 antibody. Researchers should perform their own validation if extending to these methods, following protocols similar to those established for other zebrafish protein antibodies .

How should fixation protocols be optimized when using si:dkey-29f10.1 antibody in zebrafish tissues?

When working with zebrafish tissues and the si:dkey-29f10.1 antibody, fixation protocol optimization is critical to successful detection. Based on established protocols for zebrafish antibodies:

• Fixative selection: For antibodies with unknown fixation sensitivity, test both paraformaldehyde (PFA, 4%) and formalin-based fixatives with varied fixation times (30 minutes to 2 hours)

• Epitope sensitivity assessment: Similar to other antibodies studied in zebrafish, assess whether the epitope recognized by si:dkey-29f10.1 antibody is fixation-sensitive through comparative analysis of fixed and unfixed samples

• Fixation duration: Begin with shorter fixation times (30-60 minutes) for embryonic tissues and longer times (1-2 hours) for adult tissues

• Temperature considerations: Perform fixation at room temperature initially, then test 4°C for potentially improved epitope preservation

Some antibodies against zebrafish proteins have shown significant loss of immunoreactivity upon prolonged formalin fixation, with certain conformational epitopes being particularly sensitive . Without specific published data on si:dkey-29f10.1 antibody fixation sensitivity, researchers should empirically determine optimal conditions.

What are the recommended blocking and incubation conditions for si:dkey-29f10.1 antibody in immunoassays?

For optimal results with si:dkey-29f10.1 antibody in zebrafish samples:

Western blotting conditions:

• Blocking: 5% non-fat milk or 3-5% BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature

• Primary antibody dilution: Start with 1:500-1:2000 dilution in blocking buffer

• Incubation: Overnight at 4°C with gentle agitation

• Washing: 3-5 washes with TBST, 5-10 minutes each

• Secondary antibody: HRP-conjugated anti-species antibody at 1:5000-1:10000 dilution

ELISA conditions:

• Coating: 1-10 μg/ml of capture antibody in carbonate buffer (pH 9.6)

• Blocking: 1-3% BSA in PBS for 1-2 hours at room temperature

• Sample incubation: 1-2 hours at room temperature or overnight at 4°C

• Detection antibody: 0.5-2 μg/ml of detection antibody for 1-2 hours

• Substrate: TMB with reaction stopped using 2N H₂SO₄

These recommendations are based on general protocols for zebrafish antibodies and should be optimized specifically for si:dkey-29f10.1 antibody through titration experiments .

How can si:dkey-29f10.1 antibody be used for developmental expression pattern analysis in zebrafish embryos?

For developmental expression pattern analysis using si:dkey-29f10.1 antibody in zebrafish embryos:

• Staged collection protocol:

  • Collect embryos at key developmental timepoints (e.g., 24, 48, 72, 96 hpf)

  • Process samples in parallel for comparative analysis

• Whole-mount immunostaining methodology:

  • Fix embryos in 4% PFA for 2-4 hours at room temperature or overnight at 4°C

  • Permeabilize with 0.5% Triton X-100 in PBS for 30 minutes

  • Block with 10% normal goat serum, 1% DMSO, 0.1% Tween-20 in PBS for 1-2 hours

  • Incubate with si:dkey-29f10.1 antibody (1:200-1:500) for 1-2 days at 4°C

  • Wash extensively (6-8 times, 15-30 minutes each) with PBT (PBS + 0.1% Tween-20)

  • Incubate with fluorescent secondary antibody overnight at 4°C

  • Counterstain with DAPI to visualize nuclei

• Image acquisition strategy:

  • Use confocal microscopy for high-resolution imaging

  • Collect z-stacks through the entire embryo or tissue of interest

  • Analyze expression patterns across developmental stages

This approach can be combined with in situ hybridization for si:dkey-29f10.1 mRNA to correlate protein and transcript expression patterns, similar to methods demonstrated for other zebrafish proteins .

What controls are essential when validating si:dkey-29f10.1 antibody specificity in zebrafish?

When validating si:dkey-29f10.1 antibody specificity, the following controls are essential:

• Negative controls:

  • Secondary antibody only (omitting primary antibody)

  • Pre-immune serum at the same concentration as the antibody

  • Unrelated antibody of the same isotype and concentration

  • Non-expressing tissue samples (if known)

• Peptide competition assay:

  • Pre-incubate antibody with excess purified si:dkey-29f10.1 protein or immunizing peptide

  • Compare staining between blocked and unblocked antibody

  • Specific staining should be abolished or significantly reduced

• Genetic validation:

  • Use CRISPR/Cas9 or morpholino knockdown of si:dkey-29f10.1

  • Compare antibody staining in wild-type versus knockdown samples

  • Specific staining should be reduced or absent in knockdown samples

• Molecular weight verification:

  • Verify that Western blot detection shows a band of the expected molecular weight

  • Check for cross-reactivity with other proteins

• Multi-antibody comparison:

  • If available, compare staining patterns with a second antibody raised against a different epitope of si:dkey-29f10.1

These controls are essential to establish confidence in the specificity of the antibody and rule out potential artifacts or cross-reactivity .

How can background staining be reduced when using si:dkey-29f10.1 antibody in zebrafish samples?

Background staining can be problematic when using antibodies in zebrafish tissues. To reduce background with si:dkey-29f10.1 antibody:

• Pre-absorption strategy:

  • Incubate antibody with acetone powder from non-expressing tissue

  • Centrifuge to remove bound antibodies and use supernatant

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, fish gelatin)

  • Increase blocking time (up to overnight at 4°C)

  • Use 5-10% normal serum from the species of the secondary antibody

• Antibody dilution optimization:

  • Test serial dilutions to find optimal signal-to-noise ratio

  • Consider longer incubation at higher dilution

• Washing protocol enhancement:

  • Increase number and duration of washes

  • Add 0.2-0.5M NaCl to wash buffer to reduce non-specific ionic interactions

  • Include 0.1-0.3% Triton X-100 in wash buffer for better penetration

• Autofluorescence reduction:

  • For fluorescent detection, treat with 0.1-1% sodium borohydride for 5-10 minutes

  • Include 0.1-0.3% Sudan Black B in 70% ethanol for 10-30 minutes after secondary antibody incubation

These approaches have been successfully used with other zebrafish antibodies and may help optimize si:dkey-29f10.1 antibody performance .

What steps should be taken when si:dkey-29f10.1 antibody shows cross-reactivity with off-target proteins?

If cross-reactivity is observed with si:dkey-29f10.1 antibody:

• Cross-reactivity assessment:

  • Perform Western blot analysis under various denaturing conditions

  • Compare observed bands with predicted molecular weight

  • Sequence additional bands to identify cross-reactive proteins

• Epitope analysis:

  • Use bioinformatics to identify proteins with similar epitopes

  • Test predictions with competition assays using synthetic peptides

• Antibody purification:

  • Perform affinity purification using the specific antigen

  • Use pre-adsorption against tissues from knockout models

• Alternative detection strategy:

  • Consider epitope-tagged transgenic approaches

  • Use RNA detection methods (in situ hybridization) in parallel

• Validation with genetic approaches:

  • Generate CRISPR/Cas9 knockout controls

  • Use morpholino knockdown with appropriate controls

  • Verify specificity through rescue experiments

Cross-reactivity is a common challenge with antibodies in zebrafish due to genome duplication events and paralogous genes. Careful validation is essential to distinguish between specific and non-specific signals .

How can si:dkey-29f10.1 antibody be integrated with other techniques for functional characterization?

For comprehensive functional characterization utilizing si:dkey-29f10.1 antibody:

• Integrated multi-omics approach:

  • Correlate protein expression (antibody-based) with transcriptomic data

  • Identify co-expressed genes through parallel RNA-seq analysis

  • Create protein interaction networks using co-immunoprecipitation with si:dkey-29f10.1 antibody

• Temporal-spatial expression mapping:

  • Combine antibody staining with transgenic reporter lines

  • Map expression to specific cell types using dual immunofluorescence

  • Create comprehensive developmental expression atlases

• Functional perturbation studies:

  • Use antibody to validate successful CRISPR/Cas9 or morpholino knockdown

  • Perform phenotypic analysis of knockdown embryos

  • Rescue experiments with mRNA injection followed by antibody validation

• Subcellular localization analysis:

  • Combine si:dkey-29f10.1 antibody with organelle markers

  • Use super-resolution microscopy for detailed localization

  • Perform cellular fractionation followed by Western blotting

This integrated approach provides multiple lines of evidence for protein function and helps place si:dkey-29f10.1 in its biological context .

What are the considerations for comparing si:dkey-29f10.1 expression across different experimental conditions and genetic backgrounds?

When comparing si:dkey-29f10.1 expression across different conditions:

• Standardization protocol:

  • Process all samples simultaneously with identical protocols

  • Include internal loading controls (e.g., β-actin, GAPDH)

  • Use quantitative analysis methods with appropriate normalization

• Quantification methodology:

  • For Western blots, use densitometry with linear range validation

  • For immunofluorescence, establish consistent imaging parameters

  • Consider fluorescence intensity quantification with appropriate background subtraction

• Statistical analysis approach:

  • Perform experiments with sufficient biological replicates (minimum n=3)

  • Apply appropriate statistical tests for data distribution

  • Consider power analysis to determine sample size requirements

• Genetic background considerations:

  • Account for strain-specific variations

  • Include wild-type controls from the same genetic background

  • Consider using isogenic lines for reduced variability

• Environmental variable control:

  • Standardize rearing conditions (temperature, light cycles, density)

  • Control for developmental staging (somite number, hours post-fertilization)

  • Document and control feeding regimens for post-embryonic studies

Proper experimental design and controls are essential for meaningful comparisons of si:dkey-29f10.1 expression across different conditions, particularly given the limited characterization of this protein .