si:dkey-97o5.1 Antibody

What is si:dkey-97o5.1 and what is its function in zebrafish?

Si:dkey-97o5.1 is a zebrafish gene now known as "firrm" (fignl1 interacting regulator of recombination and mitosis). This gene is predicted to enable protein kinase binding activity and is involved in interstrand cross-link repair and mitotic cell cycle regulation . According to correlation data, si:dkey-97o5.1 expression shows positive correlations with several cell cycle and DNA replication genes including pcna (r=0.118), mcm7 (r=0.107), and fen1 (r=0.103) . The gene is predicted to be localized in multiple cellular components including kinetochore, midbody, and spindle midzone .

How does si:dkey-97o5.1 relate to the human ortholog FIRRM?

Si:dkey-97o5.1 (firrm) is orthologous to human FIRRM (FIGNL1 interacting regulator of recombination and mitosis), previously known as C1orf112 . The human FIRRM protein regulates PLK1 kinase activity at kinetochores and promotes faithful chromosome segregation during prometaphase. Phosphorylation of FIRRM by PLK1 negatively regulates its interaction with the phosphatase PPP1CC, creating a negative feedback loop for maintaining proper PLK1 kinase activity during mitosis . In complex with FIGL1, FIRRM may also regulate homologous recombination . Human diseases associated with FIRRM include Adiaspiromycosis .

What techniques are recommended for detecting si:dkey-97o5.1 expression in zebrafish samples?

For detecting si:dkey-97o5.1 expression in zebrafish samples, several techniques are applicable:

• RT-PCR/qPCR: For quantitative measurement of gene expression levels

• RNA-seq: For comprehensive transcriptomic profiling

• Western blotting: For protein detection using specific antibodies

• Immunohistochemistry/Immunofluorescence: For tissue localization studies

When using antibody-based methods, validation is critical as zebrafish-specific antibodies may be limited. Cross-reactivity testing with related proteins (such as those from the si:dkey-97o5 family, including si:dkey-97o5.3 and si:dkey-97o5.5) should be performed to ensure specificity .

What is the gene expression correlation pattern of si:dkey-97o5.1 in zebrafish?

Si:dkey-97o5.1 shows distinct correlation patterns with other genes in zebrafish, as shown in the following table:

These correlations suggest si:dkey-97o5.1 is coexpressed with genes involved in DNA replication and cell cycle (pcna, mcm2-7 family, fen1), while showing negative correlations with hemoglobin genes (hbbe1.1, hbbe1.2, hbbe1.3) . This pattern supports its role in cell proliferation processes.

What are key considerations for validating a si:dkey-97o5.1 antibody for zebrafish research?

When validating a si:dkey-97o5.1 antibody for zebrafish research, consider:

• Specificity testing:

  • Western blot analysis using recombinant si:dkey-97o5.1 protein

  • Testing in si:dkey-97o5.1 knockout/knockdown models

  • Testing in tissues with known expression patterns

  • Cross-reactivity assessment with related proteins like other si:dkey-97o5 family members

• Sensitivity assessment:

  • Titration experiments to determine optimal concentrations

  • Signal-to-noise ratio evaluation in relevant tissues

• Application-specific validation:

  • Separate validation for different applications (WB, IHC, IF, IP)

  • Different fixation methods for IHC/IF applications

• Controls:

  • Positive controls (tissues with known high expression)

  • Negative controls (tissues with low/no expression)

  • Peptide competition assays to confirm epitope specificity

Similar approaches used for validating antibodies to other zebrafish proteins like prg4b can serve as methodological templates .

How should one interpret si:dkey-97o5.1 expression data in developmental studies?

Interpretation of si:dkey-97o5.1 expression during zebrafish development requires consideration of:

• Temporal expression patterns:

  • Compare expression across developmental stages

  • Relate timing to specific developmental events

  • Analyze in context of cell cycle regulation

• Spatial expression patterns:

  • Identify tissue-specific expression

  • Analyze subcellular localization (expected in kinetochore, midbody, spindle midzone)

  • Compare with expression patterns of interacting proteins

• Correlation analysis:

  • Consider co-expression with positively correlated genes (pcna, mcm family)

  • Evaluate inverse relationships with negatively correlated genes (hemoglobin family)

  • Analyze correlation changes across developmental stages

• Functional context:

  • Interpret in light of mitotic processes and DNA repair functions

  • Consider relationship to orthologous gene functions in other species

What methodological approaches are recommended for studying si:dkey-97o5.1 protein interactions?

For investigating si:dkey-97o5.1 protein interactions in zebrafish, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use validated si:dkey-97o5.1 antibodies to pull down protein complexes

  • Identify interacting partners via mass spectrometry

  • Verify specific interactions with candidate proteins through reverse Co-IP

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions in situ

  • Particularly useful for studying kinetochore and mitotic spindle interactions

• Yeast two-hybrid screening:

  • Identify novel interaction partners

  • Map interaction domains within the protein

• FRET/BRET analysis:

  • Study dynamic interactions in living cells

  • Requires fluorescent protein tagging of si:dkey-97o5.1

• Bimolecular fluorescence complementation (BiFC):

  • Visualize interactions in cellular contexts

  • Useful for localizing interactions to specific subcellular compartments

Based on human FIRRM studies, focus on interactions with PLK1 and phosphatases like PPP1CC, as these interactions are critical for its function in mitotic regulation .

How does si:dkey-97o5.1 function compare between zebrafish and human systems?

Comparing si:dkey-97o5.1 (firrm) in zebrafish to its human ortholog FIRRM reveals:

The conservation of localization and predicted functions suggests that research findings in zebrafish models may translate to human systems, particularly in cell cycle regulation contexts.

What strategies are effective for developing custom antibodies against si:dkey-97o5.1?

For developing custom antibodies against si:dkey-97o5.1, consider these research-optimized strategies:

• Epitope selection:

  • Analyze protein sequence for unique, accessible, and immunogenic regions

  • Avoid domains shared with other si:dkey-97o5 family members

  • Consider using epitope prediction algorithms

  • Target multiple epitopes distributed across the protein

• Antibody format selection:

  • Polyclonal antibodies: broader epitope recognition, useful for initial characterization

  • Monoclonal antibodies: higher specificity, better reproducibility

  • Recombinant antibodies: consistent production, potential for engineering

• Immunization and screening protocols:

  • Use multiple immunization strategies (peptide vs. recombinant protein)

  • Screen against both immunogen and full-length protein

  • Perform cross-reactivity testing against related zebrafish proteins

  • Include functional validation in relevant zebrafish tissues

• Validation criteria:

  • Western blot showing bands of expected molecular weight

  • Immunoprecipitation of the target protein

  • Signal reduction/elimination in knockdown/knockout samples

  • Subcellular localization consistent with predicted locations (kinetochore, midbody)

For zebrafish-specific applications, consider the validation approaches used for other zebrafish nuclear proteins like tprb .

How can one resolve contradictory data when characterizing si:dkey-97o5.1 antibodies?

When facing contradictory data in si:dkey-97o5.1 antibody characterization, implement this systematic resolution approach:

• Technical variability assessment:

  • Review fixation methods and their impact on epitope accessibility

  • Evaluate antibody concentration effects on signal-to-noise ratios

  • Compare detection methods (chemiluminescence vs. fluorescence)

  • Assess batch-to-batch antibody variation

• Experimental design review:

  • Verify proper controls (positive, negative, isotype)

  • Confirm target protein expression in test samples

  • Evaluate cross-reactivity with similar proteins (si:dkey-97o5.3, si:dkey-97o5.5)

  • Review sample preparation protocols

• Biological context consideration:

  • Assess developmental stage differences

  • Consider tissue-specific expression patterns

  • Evaluate post-translational modifications affecting epitope recognition

  • Review correlation data with other genes to confirm biological context

• Orthogonal method validation:

  • Compare antibody results with mRNA expression data

  • Utilize tagged protein expression for validation

  • Apply multiple antibodies targeting different epitopes

  • Implement genetic approaches (CRISPR/morpholino knockdown)

When analyzing contradictory results, prioritize functional validation in the context of si:dkey-97o5.1's predicted role in mitotic regulation and DNA repair .

What are optimal experimental designs for studying si:dkey-97o5.1 in zebrafish disease models?

For studying si:dkey-97o5.1 in zebrafish disease models, implement these optimized experimental designs:

• Genetic manipulation approaches:

  • CRISPR/Cas9 knockout or knockin models

  • Morpholino-based transient knockdown

  • Transgenic overexpression models

  • Domain-specific mutations to disrupt specific functions

• Disease-relevant phenotypic analyses:

  • Cell cycle progression assessment

  • Chromosome segregation analysis

  • DNA damage response evaluation

  • Tissue-specific proliferation measurements

• Molecular and cellular readouts:

  • Immunohistochemistry with validated antibodies

  • Live imaging with fluorescently tagged proteins

  • Gene expression analysis of correlated genes (pcna, mcm family)

  • Protein interaction studies in disease contexts

• Experimental controls and variables:

  • Include wild-type controls matched for genetic background

  • Use multiple independent mutant/transgenic lines

  • Test across different developmental stages

  • Evaluate in multiple tissue contexts

• Translational components:

  • Compare to human FIRRM function in equivalent disease models

  • Assess conservation of interactions with PLK1 and PPP1CC

  • Evaluate potential for therapeutic targeting

Given si:dkey-97o5.1's correlation with proliferation markers and predicted role in mitotic regulation , focus on cancer models and developmental disorders where cell cycle dysregulation is implicated.

What methodological approaches can resolve challenges in detecting low-abundance si:dkey-97o5.1 protein?

To overcome challenges in detecting low-abundance si:dkey-97o5.1 protein, implement these advanced methodological approaches:

• Sample enrichment strategies:

  • Subcellular fractionation targeting nuclear/kinetochore components

  • Immunoprecipitation before Western blotting

  • Density gradient ultracentrifugation

  • Cell cycle synchronization to capture peak expression phases

• Signal amplification methods:

  • Tyramide signal amplification for immunohistochemistry

  • Polymer-based detection systems

  • Proximity ligation assay for in situ detection

  • Multiple epitope targeting with antibody cocktails

• Advanced detection platforms:

  • Highly-sensitive nano-immunoassay (Simple Western)

  • Single-molecule detection methods

  • Mass spectrometry with targeted multiple reaction monitoring

  • Ultrasensitive ELISA formats with optimized antibody pairs

• Protocol optimization parameters:

  • Extended antibody incubation times at lower temperatures

  • Optimized blocking conditions to reduce background

  • Alternative detergents for improved epitope accessibility

  • Enhanced antigen retrieval methods for fixed tissues

These approaches should be calibrated using samples with known expression levels of si:dkey-97o5.1, potentially identified through the gene correlation data available from zebrafish expression databases .

How should researchers interpret differences between si:dkey-97o5.1 antibody performance in different applications?

When interpreting differences in si:dkey-97o5.1 antibody performance across applications:

• Application-specific considerations:

  • Western blot: Denatured epitopes vs. native conformation in IP/IHC

  • Immunohistochemistry: Fixation effects on epitope accessibility

  • Immunoprecipitation: Epitope masking by protein interactions

  • Flow cytometry: Surface accessibility requirements

• Technical parameter analysis:

  • Buffer composition effects on antibody binding

  • Incubation time and temperature optimization

  • Detergent type and concentration adjustments

  • Blocking reagent compatibility

• Epitope-specific factors:

  • Conformational vs. linear epitope recognition

  • Post-translational modification interference

  • Protein isoform specificity

  • Species cross-reactivity limitations

• Validation framework:

  • Use orthogonal methods to confirm findings

  • Implement application-specific positive controls

  • Test multiple antibody clones/lots

  • Perform epitope mapping to understand binding characteristics

Understanding these application-specific differences is essential for accurate data interpretation and experimental planning.

What are effective strategies for multiplexing si:dkey-97o5.1 detection with other cell cycle markers?

For effective multiplexing of si:dkey-97o5.1 with other cell cycle markers:

• Antibody selection criteria:

  • Select antibodies raised in different host species

  • Choose antibodies with complementary isotypes for secondary detection

  • Validate each antibody individually before multiplexing

  • Test for cross-reactivity between detection systems

• Sequential staining protocols:

  • Design multi-round staining with complete elution between rounds

  • Implement tyramide signal amplification for spectral separation

  • Use microwave treatment for antibody stripping between stainings

  • Document signal intensity before and after each round

• Optimal marker combinations:

  • Combine with positively correlated cell cycle markers (pcna, mcm7)

  • Include PLK1 as a functionally related marker

  • Add phase-specific cell cycle markers (cyclins, CDKs)

  • Include DNA damage markers when studying repair functions

• Advanced imaging approaches:

  • Multispectral imaging for enhanced signal separation

  • Confocal microscopy with spectral unmixing

  • Super-resolution microscopy for co-localization studies

  • Quantitative image analysis for expression correlation

This multiplexing approach can provide valuable insights into si:dkey-97o5.1's functional relationships with cell cycle regulators identified in correlation analyses .

How can researchers distinguish between si:dkey-97o5.1 and related family members in zebrafish?

To distinguish between si:dkey-97o5.1 and related family members in zebrafish:

• Sequence-based targeting:

  • Design primers/probes targeting unique regions for qPCR

  • Develop antibodies against non-conserved epitopes

  • Use RNAscope probes for highly specific in situ hybridization

  • Implement CRISPR-based tagging of endogenous proteins

• Expression pattern differentiation:

  • Compare known expression patterns (si:dkey-97o5.1 vs. si:dkey-97o5.3/prg4b vs. si:dkey-97o5.5/tprb)

  • Evaluate tissue-specific expression differences

  • Analyze developmental timing of expression

  • Compare correlation patterns with other genes

• Functional validation approaches:

  • Perform rescue experiments with specific family members

  • Use gene-specific knockdowns to observe differential phenotypes

  • Analyze subcellular localization differences (nuclear pore for tprb vs. kinetochore for firrm )

  • Evaluate protein interaction partners specific to each family member

• Cross-reactivity assessment:

  • Test antibodies against recombinant proteins of each family member

  • Perform peptide competition assays with specific epitopes

  • Use knockout/knockdown models of each gene for validation

  • Implement Western blot analysis to distinguish by molecular weight