Recombinant Blaberus craniifer Cuticle protein 9
Product Specs
Q&A
What is the molecular structure of Recombinant Blaberus craniifer Cuticle protein 9?
Recombinant Blaberus craniifer Cuticle protein 9 (also known as BcNCP3.8) is a cuticular protein with a sequence length of 34 amino acids. The complete amino acid sequence is "AVVPASTVKT ALAYTYPIHP YHSVYAHPHS VVIY" . The protein is typically expressed in E. coli expression systems for research purposes, with a purity of >85% as determined by SDS-PAGE analysis . Unlike some other cuticular proteins that contain multiple repeated motifs, the compact structure of Cuticle protein 9 suggests a specialized function within the insect exoskeleton.
How does Cuticle protein 9 compare to other cuticular proteins isolated from Blaberus craniifer?
Blaberus craniifer produces multiple cuticular proteins with varying structures and functions. According to comparative studies, Cuticle protein 9 (BcNCP3.8) is relatively small at 34 amino acids compared to Cuticle protein 8 (BcNCP21.1), which consists of 195 amino acids . Research on cuticular proteins from Blaberus craniifer has identified at least seven different nymphal endocuticular proteins that have been purified to near homogeneity . Some proteins, like Bc-NCP1, contain repeated motifs (a 16-residue motif repeated three times) with disulfide bridges, while others like Bc-NCP4 have an unusually high valine content (22.0%) . Unlike these larger, more complex proteins, Cuticle protein 9's compact structure suggests a different functional role in cuticle formation.
What methodology should be employed for proper storage and handling of Recombinant Blaberus craniifer Cuticle protein 9?
For optimal research outcomes, Recombinant Blaberus craniifer Cuticle protein 9 requires specific storage and handling protocols:
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Storage conditions: Store at -20°C for regular use or at -80°C for extended storage periods .
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Reconstitution protocol:
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Working conditions: For short-term experiments, working aliquots can be stored at 4°C for up to one week .
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Freeze-thaw cycles: Repeated freezing and thawing is not recommended as it may compromise protein integrity .
What are effective methods to study the expression patterns of Cuticle protein 9 in different developmental stages?
Studying expression patterns of Cuticle protein 9 requires a multifaceted methodological approach:
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Transcriptomic analysis: Next-generation sequencing techniques like Illumina sequencing combined with de novo assembly can characterize transcriptome-wide expression patterns. Similar approaches have been used successfully for cuticular proteins in Locusta migratoria, revealing tissue-specific and developmental stage-specific expression patterns .
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Reverse-transcription PCR (RT-PCR) and quantitative PCR (RT-qPCR): These techniques allow researchers to precisely measure expression levels across different developmental stages and tissues, as demonstrated in studies of cuticular proteins in other insect species .
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Two-dimensional gel-electrophoresis: This technique has been effective in comparing extractable proteins from selected cuticular regions of nymphs and adults of Blaberus craniifer, allowing researchers to identify stage-specific expression patterns .
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Immunohistochemistry: For precise localization studies, immunohistochemical approaches using antibodies specific to Cuticle protein 9 can reveal spatial distribution within cuticle structures, similar to approaches used for TcCPR4 protein in Tribolium castaneum .
How can researchers effectively use RNA interference to study the functional role of Cuticle protein 9?
RNA interference (RNAi) represents a powerful technique for functional characterization of Cuticle protein 9. Based on successful RNAi approaches with other cuticular proteins, researchers should consider:
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Design of target-specific dsRNA: Design double-stranded RNA specifically targeting the Cuticle protein 9 mRNA sequence, ensuring specificity by checking for potential off-target effects.
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Delivery methods: For insects like Blaberus craniifer, microinjection of dsRNA directly into the hemocoel provides the most reliable delivery method. Alternative methods include feeding or topical application depending on experimental requirements.
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Phenotypic analysis: Following RNAi treatment, comprehensive phenotypic analysis should include:
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Scanning electron microscopy to examine gross morphological changes
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Transmission electron microscopy to analyze ultrastructural alterations in cuticle organization
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Mechanical testing to measure changes in cuticle strength and flexibility
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Developmental analysis to detect effects on molting and eclosion
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This approach has proven effective in similar studies, such as research on TcCPR4 in Tribolium castaneum, where RNAi resulted in abnormal morphology of pore canals with amorphous pore canal fibers in the cuticle .
What techniques are most appropriate for investigating protein-chitin interactions involving Cuticle protein 9?
Investigating protein-chitin interactions requires specialized techniques:
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Chitin-binding assays: In vitro binding assays using purified Cuticle protein 9 and chitin substrates can determine binding affinity and kinetics.
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Cross-linking studies: Chemical cross-linking followed by mass spectrometry can identify specific amino acid residues involved in the interaction with chitin.
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Mass spectrometry analysis: MS/MS analysis has been successfully employed to recover CP peptides from chitin-binding domains, confirming that protein-chitin interactions are not mediated by covalent bonds .
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Immunogold labeling and transmission electron microscopy: This combined approach allows visualization of the precise localization of proteins within the cuticle structure relative to chitin fibrils, as demonstrated in studies of other cuticular proteins .
How does Cuticle protein 9 fit into established classification systems for insect cuticular proteins?
Insect cuticular proteins are classified into several families based on sequence motifs and structural characteristics:
Based on available literature, cuticular proteins can be divided into at least 13 different families, with the CPR family being the largest . Further sequence analysis and structural characterization would be necessary to definitively classify Cuticle protein 9 within this established framework.
What methodological approaches can effectively distinguish between the expression of Cuticle protein 9 in different cuticular structures?
Distinguishing expression patterns across cuticular structures requires specialized approaches:
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Structure-specific protein extraction: Selective extraction protocols can isolate proteins from specific cuticular regions, as demonstrated in studies comparing proteins from nymphal and adult pre-ecdysial cuticles (presumptive exocuticle) versus nymphal mid-instar cuticle (mainly endocuticle) in Blaberus craniifer .
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Immunohistochemistry with confocal microscopy: This approach can precisely localize proteins within different cuticular layers and structures.
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MS/MS analysis on isolated cuticular structures: Mass spectrometry has successfully identified cuticular proteins in different structures of Anopheles gambiae, including Johnston's organs, male antennae, eye lenses, legs, and wings .
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Quantitative analysis using normalized spectral counts: This technique allows researchers to determine relative abundance of specific proteins across different structures, revealing that typically only a few cuticular proteins are abundant in each structure .
How can researchers utilize structural data from Cuticle protein 9 to understand biomechanical properties of insect cuticle?
Understanding the relationship between Cuticle protein 9 structure and cuticle biomechanics requires:
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Mechanical testing of native versus RNAi-depleted cuticles: Comparing mechanical properties (elasticity, hardness, fracture resistance) between normal cuticles and those with reduced Cuticle protein 9 can reveal functional contributions.
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Structure-function correlation studies: Analyzing how specific domains or residues within Cuticle protein 9 contribute to cuticle properties through site-directed mutagenesis.
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Computational modeling: Molecular dynamics simulations can predict how Cuticle protein 9 interactions with chitin and other cuticular proteins influence mechanical properties at the molecular level.
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Atomic force microscopy (AFM) analysis: AFM can measure nanoscale mechanical properties of cuticle samples with and without Cuticle protein 9, providing direct evidence of its mechanical contribution.
Similar studies with TcCPR4 in Tribolium castaneum demonstrated that this protein is required for proper morphology of vertical pore canals and pore canal fibers that contribute to cuticle rigidity while maintaining lightweight properties .
What methodological approaches can effectively analyze the interaction between Cuticle protein 9 and other cuticular proteins in forming complex structures?
Analyzing protein-protein interactions in cuticular assemblies requires:
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Co-immunoprecipitation studies: Identify protein interaction partners of Cuticle protein 9 within the cuticle complex.
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Proximity-dependent labeling techniques: Methods like BioID or APEX can identify proteins in close proximity to Cuticle protein 9 in vivo.
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Two-hybrid screening: Identify potential binding partners through yeast or bacterial two-hybrid systems.
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Cross-linking mass spectrometry: Identify specific residues involved in interactions between Cuticle protein 9 and other cuticular components.
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Correlative microscopy approaches: Combine immunolocalization with electron microscopy to visualize spatial relationships between different proteins within cuticle structures.
Research on other cuticular proteins suggests complex interactions between multiple proteins and chitin in forming functional cuticle structures, with specific proteins being localized to particular regions like pore canals .
What methodological approaches can effectively trace the evolutionary history of Cuticle protein 9 across insect lineages?
Evolutionary analysis of Cuticle protein 9 requires:
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Phylogenetic analysis: Construct phylogenetic trees using Cuticle protein 9 sequences from diverse insect species to trace evolutionary relationships.
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Sequence conservation analysis: Identify highly conserved regions that may be functionally important across species.
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Synteny analysis: Compare genomic organization of the Cuticle protein 9 gene and surrounding regions across species to identify evolutionary patterns.
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Selection pressure analysis: Calculate dN/dS ratios to determine whether Cuticle protein 9 has been under purifying, neutral, or positive selection.
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Ancestral sequence reconstruction: Predict ancestral sequences to understand the evolutionary trajectory of Cuticle protein 9.
These approaches could reveal whether Cuticle protein 9 represents a conserved ancient protein or a more recent evolutionary innovation within specific insect lineages.
How do expression patterns of Cuticle protein 9 homologs differ between rigid and flexible cuticle structures across insect species?
Understanding differential expression patterns requires comparative approaches:
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Transcriptomic comparison: Compare expression levels of Cuticle protein 9 homologs in rigid versus flexible cuticles across multiple species.
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Immunohistochemical localization: Use antibodies against conserved epitopes to compare localization patterns in different cuticle types.
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Laser capture microdissection: Isolate specific cuticular regions for targeted transcriptomic or proteomic analysis.
Studies in Tribolium castaneum have shown that some cuticular proteins like TcCPR4 are present in rigid cuticles (dorsal elytron, ventral abdomen, leg) but absent in flexible cuticles (hindwing, dorsal abdomen) . Similar comparative analyses could reveal whether Cuticle protein 9 shows specialized distribution patterns corresponding to mechanical properties of different cuticular structures.
