si:dkey-18l1.1 Antibody

Advanced Research Questions

• How should researchers validate a new si:dkey-18l1.1/vwa8 antibody for zebrafish research?

Antibody validation is critical for ensuring reproducibility in research. For si:dkey-18l1.1/vwa8 antibodies, validation should include:

• Specificity testing:

  • Western blot to confirm single band of expected molecular weight (~214.8 kDa for VWA8)

  • Testing in vwa8 knockout or knockdown samples as negative controls

  • Peptide competition assays to confirm epitope specificity

• Sensitivity assessment:

  • Titration experiments to determine optimal working concentration

  • Detection limits in samples with varying expression levels

• Application-specific validation:

  • For immunohistochemistry: confirmation of expected tissue distribution pattern

  • For immunoprecipitation: confirmation of successful pull-down with mass spectrometry

• Cross-reactivity testing:

  • Testing on closely related proteins

  • Confirmation of specificity across developmental stages

• Reproducibility assessment:

  • Testing across multiple batches of antibody

  • Testing by multiple researchers

Each validation approach contributes to building confidence in antibody performance and specificity, which is essential for generating reliable research data .

• What experimental approaches can be used to study si:dkey-18l1.1/vwa8 function in notochord development?

Since vwa8 is predicted to act upstream of or within notochord development , several methodological approaches can be employed:

• Spatiotemporal expression analysis:

  • Immunohistochemistry with si:dkey-18l1.1/vwa8 antibodies during developmental stages

  • Co-staining with notochord markers to determine precise localization

• Loss-of-function studies:

  • CRISPR-Cas9 knockout of vwa8

  • Morpholino-mediated knockdown

  • Documenting phenotypic effects on notochord formation and function

• Domain-function analysis:

  • Creating mutations in specific domains (e.g., von Willebrand factor A domains or AAA+ ATPase domains)

  • Testing which functional aspects of the protein are essential for notochord development

• Protein interaction mapping:

  • Immunoprecipitation with si:dkey-18l1.1/vwa8 antibodies followed by mass spectrometry

  • Identifying interaction partners in the notochord development pathway

• Live imaging:

  • Tagging vwa8 with fluorescent proteins

  • Time-lapse imaging during notochord development

  • Correlating with antibody staining to validate tag functionality

• Rescue experiments:

  • Reintroducing wild-type or mutant vwa8 into knockouts

  • Testing which forms restore normal notochord development

These approaches would provide mechanistic insights into how vwa8 contributes to notochord formation in zebrafish .

• How can si:dkey-18l1.1/vwa8 antibodies be applied in studying potential links to human retinitis pigmentosa?

Zebrafish vwa8 has been identified as orthologous to human VWA8, which has associations with retinitis pigmentosa 97 . This connection enables several research approaches:

• Comparative expression analysis:

  • Using si:dkey-18l1.1/vwa8 antibodies to map protein expression in zebrafish retina

  • Comparing patterns with human VWA8 expression in normal and diseased retinal tissues

• Disease modeling:

  • Creating zebrafish with mutations mimicking those found in human retinitis pigmentosa

  • Using antibodies to track changes in protein expression, localization, or interactions

  • Analyzing retinal degeneration phenotypes

• Functional studies:

  • Examining vwa8 function in zebrafish retinal development and maintenance

  • Correlating with potential mechanisms in human disease

  • Documenting phenotypes in visual function tests

• Drug screening:

  • Using zebrafish vwa8 models and antibody-based assays to screen compounds

  • Identifying molecules that might restore normal protein function or expression

  • Testing promising candidates in more advanced models

• Mitochondrial function assessment:

  • Since vwa8 is predicted to localize to mitochondria , examining its role in retinal mitochondrial function

  • Using antibodies to track changes in protein localization under stress conditions

This translational approach leverages the experimental advantages of zebrafish while providing insights potentially relevant to human retinal disease mechanisms .

Methodological Questions

• What is the optimal protocol for Western blotting with si:dkey-18l1.1/vwa8 antibodies in zebrafish samples?

Based on available information on related antibodies and considering the properties of vwa8, the following protocol is recommended:

Sample preparation:

• Harvest appropriate tissues (liver, kidney, spleen recommended based on expression data)

• Homogenize in RIPA buffer containing protease inhibitors

• Centrifuge at 14,000×g for 15 minutes at 4°C

• Quantify protein concentration using Bradford or BCA assay

SDS-PAGE:

• Use 6-8% gels (optimal for large proteins like vwa8, ~214.8 kDa)

• Load 20-50 μg protein per lane

• Include molecular weight markers covering high molecular weight range

Transfer:

• Transfer to PVDF membrane (preferred for high molecular weight proteins)

• Use wet transfer at 30V overnight at 4°C for efficient transfer of large proteins

• Verify transfer with reversible stain (e.g., Ponceau S)

Immunoblotting:

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

• Incubate with primary antibody diluted 1:500-1:1000 in blocking buffer overnight at 4°C

• Wash 3× with TBST, 10 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody (1:5000) for 1 hour

• Wash 3× with TBST, 10 minutes each

• Develop using enhanced chemiluminescence detection system

Controls:

• Positive control: Tissue with known vwa8 expression (e.g., liver)

• Negative control: Samples from vwa8 knockdown or knockout if available

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

This protocol may require optimization based on the specific antibody characteristics and sample types .

• What protein interaction methods can be combined with si:dkey-18l1.1 antibodies?

Several complementary techniques can be used with si:dkey-18l1.1/vwa8 antibodies to study protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use si:dkey-18l1.1 antibodies to precipitate vwa8 and associated proteins

  • Identify binding partners via Western blot or mass spectrometry

  • Perform reciprocal Co-IP with antibodies against suspected interacting partners

  • Example protocol parameters: 2-5 μg antibody per 500 μg protein lysate, overnight incubation at 4°C

• Proximity Ligation Assay (PLA):

  • Combine si:dkey-18l1.1 antibodies with antibodies against potential interacting partners

  • Generates fluorescent signals only when proteins are within 40 nm

  • Allows visualization of interactions directly in tissues or cells

• Immunofluorescence co-localization:

  • Double staining with si:dkey-18l1.1 antibodies and antibodies against potential partners

  • Use confocal microscopy for high-resolution imaging

  • Quantify co-localization using Pearson's correlation coefficient or Manders' overlap coefficient

• Cross-linking coupled with immunoprecipitation:

  • Stabilize transient interactions with membrane-permeable crosslinkers

  • Use si:dkey-18l1.1 antibodies to isolate crosslinked complexes

  • Identify interacting proteins by mass spectrometry

• ELISA-based interaction assays:

  • Similar to inhibitor screening ELISA kits , develop assays where:

    • Coat plates with recombinant vwa8

    • Add potential interacting proteins

    • Detect using antibodies against the interacting proteins

These methods provide complementary data on protein interactions, with each technique offering different advantages in terms of sensitivity, specificity, and ability to detect transient or weak interactions .

• How should researchers design experiments to study the mitochondrial functions of vwa8 using si:dkey-18l1.1 antibodies?

Since vwa8 is predicted to localize to mitochondria , the following experimental approaches are recommended:

• Subcellular localization confirmation:

  • Perform subcellular fractionation to isolate mitochondria

  • Western blot with si:dkey-18l1.1 antibodies to confirm presence in mitochondrial fraction

  • Compare with established mitochondrial markers (e.g., VDAC, COX IV)

  Fraction	vwa8	VDAC (mito marker)	GAPDH (cytosolic marker)
  Total lysate	+	+	+
  Cytosolic	+/-	-	+
  Mitochondrial	+	+	-

• Co-localization studies:

  • Perform immunofluorescence with si:dkey-18l1.1 antibodies and mitochondrial dyes

  • Use super-resolution microscopy for detailed localization

  • Quantify degree of co-localization under different conditions

• Mitochondrial function correlation:

  • Manipulate vwa8 expression (knockdown/overexpression)

  • Measure changes in:

    • Oxygen consumption rate

    • ATP production

    • Mitochondrial membrane potential

    • ROS production

  • Correlate functional changes with vwa8 levels detected by antibodies

• Mitochondrial dynamics:

  • Track vwa8 during mitochondrial processes (fusion, fission, mitophagy)

  • Use live-cell imaging with tagged vwa8 validated by antibody staining

  • Examine co-localization with dynamics machinery proteins

• ATPase activity studies:

  • Given the AAA+ ATPase domains in vwa8 :

    • Immunoprecipitate native vwa8 using antibodies

    • Measure ATPase activity in immunoprecipitates

    • Test effects of mitochondrial stress on activity

These approaches would provide comprehensive data on the mitochondrial functions of vwa8 in zebrafish, potentially revealing its role in mitochondrial biology and disease mechanisms .

• What technical considerations are important when using si:dkey-18l1.1 antibodies in developmental studies of zebrafish?

When studying vwa8 expression during zebrafish development, researchers should consider:

• Developmental stage selection:

  • Based on notochord development timeline

  • Include stages before, during, and after notochord formation

  • Consider time points at 24, 48, 72, and 96 hours post-fertilization

• Tissue processing optimization:

  • Fixation: Test multiple fixatives (4% PFA, Dent's fixative) for optimal epitope preservation

  • Permeabilization: Carefully optimize to maintain tissue integrity while allowing antibody access

  • Antigen retrieval: May be necessary for some fixation methods

• Background reduction strategies:

  • Use appropriate blocking (5-10% normal serum matching secondary antibody host)

  • Include detergents (0.1-0.3% Triton X-100) to reduce non-specific binding

  • Consider autofluorescence quenching for fluorescent detection

• Controls:

  • Developmental series of wild-type embryos

  • vwa8 knockdown or knockout embryos as negative controls

  • Secondary antibody-only controls

  • Pre-absorption controls with immunizing peptide

• Quantification methods:

  • Standardize image acquisition parameters

  • Use appropriate software for quantitative analysis

  • Normalize to internal controls

• Correlation with expression data:

  • Compare antibody staining patterns with mRNA expression

  • Validate findings with multiple techniques (e.g., in situ hybridization)

By addressing these considerations, researchers can generate reliable data on vwa8 expression and function during zebrafish development, particularly in relation to its predicted role in notochord development .