Recombinant Leucoraja ocellata Gap junction delta-2 protein

What is Recombinant Leucoraja ocellata Gap Junction Delta-2 protein?

Recombinant Leucoraja ocellata Gap Junction Delta-2 protein (P69999) is a full-length protein (1-302 amino acids) derived from the Winter skate (Raja ocellata), expressed in E. coli with an N-terminal His tag. It is also known as Connexin-35 (Cx35) or Gap junction alpha-9 protein, representing a major component of electrical synapses . This protein is primarily studied for its role in gap junction formation and its implications in neurological and visual system development.

What are the optimal storage conditions for Recombinant Leucoraja ocellata Gap Junction Delta-2 protein?

For optimal stability, store the protein at -20°C/-80°C upon receipt, with proper aliquoting to avoid multiple freeze-thaw cycles. The protein is supplied as a lyophilized powder in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 . For working aliquots, storage at 4°C for up to one week is recommended. After reconstitution, adding glycerol to a final concentration of 5-50% (with 50% being standard) before aliquoting provides optimal long-term stability .

What is the relationship between Leucoraja ocellata Gap Junction Delta-2 protein and other connexin orthologs?

Leucoraja ocellata Gap Junction Delta-2 protein (Cx35) is orthologous to mammalian connexin-36 (Cx36). In zebrafish, there are four Cx36 orthologs: gjd1a/Cx34.1, gjd2b/Cx35.1, gjd1b/Cx34.7, and gjd2a/Cx35.5 . These proteins form the molecular basis of electrical synapses in vertebrates, with specific distributions in neuronal tissues, particularly in the retina. The evolutionary conservation of these proteins across species makes them valuable models for understanding fundamental aspects of electrical synaptic transmission .

What is the recommended protocol for reconstituting lyophilized Recombinant Leucoraja ocellata Gap Junction Delta-2 protein?

For optimal reconstitution:

• Centrifuge the vial briefly to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• For long-term storage, add glycerol to a final concentration of 5-50% (50% is standard)

• Aliquot to avoid repeated freeze-thaw cycles

• Store reconstituted protein at -20°C/-80°C for long-term use

This protocol maintains protein stability and functionality for subsequent experimental applications.

How can researchers verify the purity and functionality of Recombinant Leucoraja ocellata Gap Junction Delta-2 protein?

To verify protein quality:

• Conduct SDS-PAGE analysis to confirm purity (should be >90% pure)

• Perform Western blot analysis using anti-His antibodies to confirm identity

• For functional verification, employ electrophysiological techniques to assess gap junction channel formation

• Utilize immunofluorescence microscopy to evaluate subcellular localization

• Assess protein-protein interactions through co-immunoprecipitation assays

These methods collectively ensure the recombinant protein maintains structural integrity and biological activity.

What considerations should be made when designing immunohistochemical experiments with Recombinant Leucoraja ocellata Gap Junction Delta-2 protein?

When conducting immunohistochemical studies:

• Be aware that antibodies against Cx35/36 may cross-react with closely related connexin orthologs (gjd1a/Cx34.1, gjd1b/Cx34.7, gjd2a/Cx35.5)

• Include appropriate controls (e.g., knockout tissue) to differentiate specific from non-specific staining

• Focus examination on known expression sites like the inner plexiform layer and between photoreceptor cells

• Use confocal microscopy for optimal resolution of gap junction plaques

• Consider double-labeling with synaptic markers to identify electrical synapses

The lack of antibodies that discriminate between the four zebrafish Cx35 orthologs presents a significant challenge, requiring careful experimental design and interpretation .

How can Recombinant Leucoraja ocellata Gap Junction Delta-2 protein be utilized in refractive error studies?

For investigating refractive development:

• Employ the protein in comparative studies with knockout models to understand its role in eye development

• Use optical coherence tomography (OCT) to measure ocular parameters including axial length

• Assess changes in Wnt/β-catenin signaling pathways, which are affected by gap junction function

• Examine connections between connexin expression and dopamine receptor activity

• Analyze visual-motor responses to light transitions for functional assessment

Research has shown that depletion of gjd2b/Cx35.1 affects eye axial length, leading to hyperopic shifts and altered visual-motor behavioral responses, suggesting a critical role in refractive development .

What molecular mechanisms might explain the role of Gap Junction Delta-2 protein in visual system development?

Current research indicates several potential mechanisms:

• Regulation of intercellular communication between photoreceptor cells, affecting visual signal processing

• Modulation of Wnt/β-catenin signaling pathways, which are critical for eye development

• Influence on expression of other connexins (gjd1b/Cx34.7, gja3/Cx48.5) involved in retinal function

• Interaction with dopaminergic signaling systems that regulate retinal activity

• Contribution to the development of neural circuits in the retina

The reduction in gjd1b/Cx34.7 expression observed in Cx35.1-depleted models suggests that heterotypic gap junctions between these connexins may be essential for normal visual function .

What are the implications of Gap Junction Delta-2 protein depletion on cone and rod photoreceptor function?

Research on Cx35.1 depletion reveals:

• Enhanced cone photoreceptor activity in response to bright light transitions

• Downregulation of rhodopsin genes, suggesting decreased rod photoreceptor sensitivity

• Reversal of visual disturbances under low/mesopic light conditions

• Alteration of visual-motor behavioral responses to abrupt light transitions

• Evidence for a cone-mediated process causing VMR light-ON hyperactivity

These findings suggest differential effects on rod and cone pathways, with particular implications for cone-dominated visual processing under photopic conditions .

How do zebrafish models complement studies of Recombinant Leucoraja ocellata Gap Junction Delta-2 protein?

Zebrafish models offer several advantages:

• High conservation of signaling pathways regulating nervous system development from fish to humans

• Accessibility to CRISPR-Cas9 genome engineering for creating knockout models

• Transparent embryos allowing direct visualization of developing structures

• Well-established behavioral assays for visual function assessment

• Ability to study developmental roles of electrical synapses in vivo

The zebrafish gjd2b/Cx35.1 knockout models have provided valuable insights into the role of this protein in eye development, refractive error, and visually guided behaviors .

What are the key differences between Gap Junction Delta-2 protein orthologs across species?

Comparative analysis reveals:

• Zebrafish possess four Cx36 orthologs (gjd1a/Cx34.1, gjd2b/Cx35.1, gjd1b/Cx34.7, gjd2a/Cx35.5) compared to a single gene in mammals

• Differential expression patterns exist across species, with tissue-specific specialization

• Functional asymmetry at electrical synapses is conserved but may be mediated by different combinations of connexins

• Species-specific roles in development, with distinct phenotypes upon gene depletion

• Varied relationships with other connexin family members that form heterotypic junctions

Understanding these differences is crucial for translating findings across model systems and interpreting the evolutionary significance of gap junction function .

How can CRISPR-Cas9 genome engineering be optimized for studying Gap Junction Delta-2 protein function?

For effective CRISPR-Cas9 application:

• Target exon one upstream of the start codon for complete functional knockout

• Screen for minimal mutations (e.g., 1bp substitution) that create early stop codons

• Verify germline transmission through multiple generations

• Confirm knockout at both RNA and protein levels

• Consider compensatory regulation of related connexin genes when interpreting phenotypes

In zebrafish studies, targeting position G12→A in exon one created an early stop codon at amino acid 4, effectively eliminating functional Cx35.1 expression while allowing assessment of compensatory mechanisms .

What are the emerging techniques for studying Gap Junction Delta-2 protein interactions in the visual system?

Cutting-edge approaches include:

• Super-resolution microscopy techniques (STORM, PALM) to visualize gap junction plaques at nanoscale resolution

• Optogenetic manipulation of connexin-expressing cells to assess functional connectivity

• Single-cell RNA sequencing to map connexin expression patterns across retinal cell types

• CRISPR-based gene editing combined with inducible systems for temporal control of gene expression

• Advanced electrophysiological techniques to measure electrical coupling between specific neuronal populations

These techniques will enable more precise understanding of the spatial and temporal dynamics of gap junction formation and function in the developing visual system.

How might Gap Junction Delta-2 protein research contribute to understanding human refractive disorders?

Translational implications include:

• Identification of genetic factors contributing to refractive error development in humans

• Understanding the molecular pathways linking electrical synapses to eye growth regulation

• Development of targeted interventions to modulate gap junction function for refractive error control

• Insights into the role of retinal signaling in emmetropization (normal eye development)

• Potential therapeutic targets for conditions like myopia and hyperopia

The conservation of connexin function across species suggests that findings from Leucoraja ocellata and zebrafish models may have direct relevance to human ocular development and refractive disorders .

What is the relationship between Gap Junction Delta-2 protein and lens development?

Emerging research indicates:

These findings suggest a previously unappreciated role for neuronal connexins in influencing lens development and function, potentially through indirect signaling mechanisms .