si:ch211-163l21.7 Antibody

What is si:ch211-163l21.7 and what cellular structures is it associated with?

Si:ch211-163l21.7 is a protein-coding gene located on chromosome 8 in zebrafish. It is predicted to be localized to axonemal microtubules and is orthologous to human CFAP276 (cilia and flagella associated protein 276). This gene belongs to the cilia- and flagella-associated protein 276 family (InterPro ID: IPR022179) and has been identified as having potential roles in ciliary and flagellar structures . Two transcript variants have been documented: an mRNA transcript (si:ch211-163l21.7-201) with a length of 1,156 nucleotides and a non-coding RNA variant (si:ch211-163l21.7-002) that is 852 nucleotides long .

How is si:ch211-163l21.7 related to mitochondrial function in zebrafish?

While si:ch211-163l21.7 is primarily associated with axonemal structures, research has identified potential relationships between this gene and mitochondrial proteins such as ES1. In zebrafish retinal tissue studies, si:ch211-163l21.7 has been mentioned in the context of mitochondrial enlargement factors, suggesting it may play a role in mitochondrial development or function, particularly in specialized cells like cone photoreceptors . Understanding this relationship requires careful immunolocalization studies to determine whether si:ch211-163l21.7 protein co-localizes with mitochondrial markers or interacts with known mitochondrial proteins.

What criteria should researchers use when selecting antibodies against si:ch211-163l21.7?

When selecting antibodies for si:ch211-163l21.7 research, prioritize antibodies raised against unique epitopes that do not cross-react with paralogues. Given the presence of related proteins in zebrafish, validation through multiple approaches is essential. Look for antibodies that have been validated through immunoblotting, immunohistochemistry, and ideally through knockout/knockdown controls. Consider the specific application requirements, including fixation compatibility, species reactivity, and whether native or denatured protein detection is needed. For colocalization studies with mitochondrial markers (such as TOM20), ensure the primary antibodies are raised in different species to allow simultaneous detection .

What are the recommended validation steps for si:ch211-163l21.7 antibodies?

Thorough validation should include:

• Immunoblotting to confirm specificity (single band of expected molecular weight)

• Immunoprecipitation followed by mass spectrometry

• Comparing staining patterns with transcript localization (in situ hybridization)

• Demonstrating reduced or absent signal in knockdown or knockout models

• Peptide competition assays to confirm epitope specificity

Research involving zebrafish retinal tissues has demonstrated that proper antibody validation includes confirming single band detection on immunoblots corresponding to the calculated molecular mass of the target protein, as shown with other mitochondrial proteins like ES1, TOM20, and mAAT .

What are effective immunohistochemistry protocols for si:ch211-163l21.7 detection in zebrafish tissues?

For optimal detection of si:ch211-163l21.7 in zebrafish tissues, particularly retina, researchers should consider a protocol similar to that used for related proteins:

• Fix tissues in 4% paraformaldehyde in phosphate buffer (4-24 hours depending on tissue size)

• Cryoprotect in 30% sucrose solution

• Embed in OCT compound and section at 10-12 μm thickness

• Block with 5% normal serum corresponding to secondary antibody species with 0.1-0.3% Triton X-100

• Incubate with primary antibody (optimized dilution, typically 1:200-1:1000) overnight at 4°C

• Wash extensively with PBS (3-5 times, 10 minutes each)

• Incubate with fluorophore-conjugated secondary antibody for 1-2 hours at room temperature

• Counterstain nuclei with DAPI if desired

• Mount in anti-fade medium

For co-localization studies, simultaneous incubation with antibodies against mitochondrial markers such as TOM20 can be performed as demonstrated in previous zebrafish retina studies .

How can subcellular fractionation optimize si:ch211-163l21.7 antibody applications?

Subcellular fractionation enhances si:ch211-163l21.7 antibody applications by:

• Separating soluble from membrane-bound proteins to determine localization

• Enriching target protein concentration for improved detection sensitivity

• Reducing background from unrelated cellular components

• Facilitating comparison between different cellular compartments

A recommended protocol based on previous research :

• Homogenize fresh tissue in isotonic buffer (250 mM sucrose, 10 mM HEPES, pH 7.4, 1 mM EDTA) with protease inhibitors

• Centrifuge at 600g for 10 minutes to remove nuclei and debris

• Centrifuge supernatant at 10,000g for 15 minutes to isolate crude mitochondria

• Further fractionate mitochondria into membranes and soluble components if needed

• Use markers such as alpha-tubulin (soluble fraction) and TOM20 (membrane fraction) as controls

This approach helps determine whether si:ch211-163l21.7 is present in the mitochondrial matrix, intermembrane space, or associated with membranes .

How can morpholino knockdown approaches be optimized for si:ch211-163l21.7 functional studies?

When designing morpholino (MO) knockdown studies for si:ch211-163l21.7:

• Design two types of morpholinos for validation purposes:

  • Splice-blocking MO to generate frame-shifted mRNA (allows PCR verification of knockdown)

  • Translation-blocking MO targeting the start codon region

• Avoid sequences highly conserved between si:ch211-163l21.7 and its paralogues to prevent off-target effects

• Validate knockdown efficiency:

  • For splice-blocking MOs, use RT-PCR to confirm altered splicing

  • For translation-blocking MOs, use immunoblotting to confirm reduced protein levels

• Include appropriate controls:

  • Standard control MO (non-targeting)

  • Rescue experiments with MO-resistant mRNA constructs

• Assess phenotypes using multiple approaches:

  • Immunohistochemistry with multiple markers (e.g., TOM20 and mAAT for mitochondria)

  • Electron microscopy to examine subcellular structures directly

This approach has been successfully used to study related proteins in zebrafish, demonstrating measurable changes in mitochondrial size and distribution .

What are the considerations for designing CRISPR/Cas9 knockout models for si:ch211-163l21.7?

When establishing CRISPR/Cas9 knockout models:

• Design multiple sgRNAs targeting early exons to maximize knockout efficiency

• Validate genomic editing using T7 endonuclease assays and sequencing

• Screen F0 mosaic founders by analyzing small tissue biopsies

• Assess potential compensatory effects from paralogues that may mask phenotypes

• Compare knockdown vs. knockout phenotypes, as they may differ due to developmental adaptation

Previous research has shown that CRISPR/Cas9-based approaches can yield different phenotypes compared to morpholino knockdowns, potentially due to genetic compensation mechanisms. For example, while MO-injected larvae showed reduced mitochondrial marker signals in cones, sgRNA-injected larvae did not demonstrate the same reduction in mitochondrial size, indicating potential compensatory mechanisms in complete knockout models .

How does zebrafish si:ch211-163l21.7 compare to human CFAP276 for antibody-based studies?

Zebrafish si:ch211-163l21.7 is orthologous to human CFAP276 (cilia and flagella associated protein 276) . When conducting comparative studies:

• Epitope conservation should be assessed when selecting antibodies:

  • Sequence alignments between zebrafish and human proteins

  • Confirmation of epitope presence in both species

  • Validation of cross-reactivity if using the same antibody

• Functional studies should address:

  • Conservation of protein-protein interactions

  • Subcellular localization similarities and differences

  • Tissue-specific expression patterns

• Consider complementary approaches:

  • Human cell lines expressing fluorescently-tagged CFAP276

  • Expression of human CFAP276 in zebrafish knockout models

This comparative approach allows researchers to leverage the advantages of zebrafish models while maintaining translational relevance to human biology.

What strategies can resolve weak or inconsistent immunostaining with si:ch211-163l21.7 antibodies?

When facing weak or inconsistent immunostaining:

• Optimize fixation protocols:

  • Test multiple fixatives (PFA, methanol, acetone)

  • Vary fixation duration (2-24 hours)

  • Consider antigen retrieval methods (heat-induced, enzymatic)

• Address antibody permeability issues:

  • Increase detergent concentration (0.1-0.5% Triton X-100)

  • Test longer permeabilization times

  • Consider freeze-thaw cycles for difficult tissues

• Signal amplification options:

  • Tyramide signal amplification

  • Multilayer detection systems

  • Extended antibody incubation times (overnight to 48 hours)

• Consider tissue-specific challenges:

  • Previous research has shown that antibody permeability issues can affect detection in zebrafish cone mega-mitochondria, resulting in weaker signals in apical and central regions of cone ellipsoids despite uniform distribution at the electron microscopy level .

How can researchers address potential cross-reactivity with paralogues?

To minimize cross-reactivity issues:

• Peptide competition assays:

  • Pre-incubate antibody with specific peptides from si:ch211-163l21.7

  • Pre-incubate with peptides from paralogues

  • Compare staining patterns to identify specific vs. cross-reactive signals

• Knockout/knockdown controls:

  • Use both si:ch211-163l21.7-specific and paralogue-specific knockdowns

  • Compare staining patterns to identify antibody specificity

• Western blot analysis with recombinant proteins:

  • Express recombinant si:ch211-163l21.7 and paralogues

  • Test antibody reactivity against each protein

This is particularly important since previous research has identified that some zebrafish proteins have paralogues with high homology that can substitute functionally in diverse tissues .

What quantitative methods are appropriate for analyzing si:ch211-163l21.7 immunostaining in zebrafish tissues?

For robust quantification of immunostaining:

• Signal intensity measurements:

  • Define regions of interest (ROIs) consistently across samples

  • Use relative signal intensity normalized to control regions

  • Present data as box-whisker plots showing median, quartiles, and range

• Colocalization analysis:

  • Calculate Pearson's or Mander's coefficients when studying potential interactions

  • Compare colocalization metrics across different experimental conditions

• Morphometric analysis for subcellular structures:

  • Outline individual organelles (e.g., mitochondria) to measure size and shape

  • Count and measure structures to determine density and distribution

Previous research used relative signal intensity quantification to compare mitochondrial marker expression between control and morpholino-injected zebrafish larvae, presenting results as box-whisker plots and setting the mean value of control classes to 1.0 for standardization .

How should researchers integrate immunofluorescence data with electron microscopy findings?

When integrating light and electron microscopy data:

• Correlative approaches:

  • Use sequential sections for immunofluorescence and electron microscopy

  • Apply immunogold electron microscopy for direct correlation

  • Document identical regions with both techniques

• Quantitative integration:

  • Measure structures at both resolution levels

  • Correlate fluorescence intensity with ultrastructural features

  • Use statistical approaches to validate relationships

• 3D reconstruction techniques:

  • Serial section electron microscopy

  • Volume electron microscopy (SBF-SEM, FIB-SEM)

  • Registration with confocal z-stacks

This integrated approach has been successfully applied in zebrafish retina research, where immunofluorescence findings of reduced mitochondrial marker signals were confirmed at the ultrastructural level, revealing smaller mitochondria in morpholino-treated animals .

How might single-cell approaches advance understanding of si:ch211-163l21.7 function?

Single-cell approaches offer several advantages:

• Cell type-specific expression analysis:

  • Single-cell RNA-seq to identify cells expressing si:ch211-163l21.7

  • Spatial transcriptomics to map expression in intact tissues

  • Integration with antibody-based protein detection

• Functional heterogeneity assessment:

  • Single-cell protein quantification

  • Correlation of expression with morphological features

  • Cell-to-cell variability analysis

• Lineage tracing combined with si:ch211-163l21.7 detection:

  • Temporal analysis of expression during development

  • Correlation with cell fate decisions

  • Identification of regulatory mechanisms

These approaches could help resolve cell-type specific functions in tissues like the zebrafish retina, where si:ch211-163l21.7 may play different roles in different photoreceptor types.

Methodological Considerations Table

How do monoclonal versus polyclonal antibodies compare for si:ch211-163l21.7 detection?

When selecting between monoclonal and polyclonal antibodies:

• Monoclonal antibodies offer:

  • High specificity for a single epitope

  • Batch-to-batch consistency

  • Reduced background in most applications

  • Potentially lower sensitivity for rare epitopes

• Polyclonal antibodies provide:

  • Recognition of multiple epitopes for enhanced signal

  • Greater tolerance to protein modifications

  • Better performance in certain applications like immunoprecipitation

  • Potentially higher background

• Application-specific considerations:

  • For novel proteins like si:ch211-163l21.7, initial characterization with polyclonal antibodies may provide broader epitope recognition

  • Follow-up studies requiring precise epitope targeting benefit from monoclonal antibodies

  • Validation should include comparing results from both antibody types when possible

The choice should be guided by the specific research question, available validation data, and the particular application requirements.