Здесь мы опишем эффективные стратегии для удаления шелкового желез из живота женщины черные вдовы. Эта процедура позволяет быстрое изоляции семь различных шелковых желез в высокой степени очистки моды, важный процесс для следователей изучения паук производства шелка и волокон сборки.
Abstract
Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.
Protocol
1. Обезболивающий паука и изоляции брюшной полости Передача паука из стеклянной банке в картонной коробке с пластиковой подкладке (рис. 1А). Как правило, мы собираем пауков от woodpiles, гаражи или кустарников и их размещение в лаборатории в стеклянных банках. Потому что робот не может п…
Discussion
Наша методология микродиссекции шелковой желез от черного паука вдовы предлагает эффективные средства для получения высокой степени очистки шелка производству желез. Вскрытия может быть завершена в 1,5 до 3 часов, что дает полный набор семь различных шелковых желез из cobweavers. Получение …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Эта работа была поддержана NSF RUI Грант MCB-0950372 озаглавленном молекулярная характеристика черного шелка паука вдовы.
Materials
Material Name
Type
Company
Catalogue Number
Comment
Sodium chloride
EM Science
SX0420-1
0.1 M in water
Diethyl pyrocarbonate
Sigma
D-5758 – 5 ml
0.1% v/v
Sodium citrate
Sigma
S1804 – 1 kg
0.015 M in water
Dissecting microscope
Leica Microsystems
Leica MZ16
Digital microscope camera
Leica Microsystems
DFC320
Software – Leica Application Suite v2.8.1
Vannas scissors
World Precision Instruments
500260
Stainless steel forceps
World Precision Instruments
501764
Mini Dumont #M5S
Insect pins
Indigo Instruments
33414-2
Insect pins #2
Small or large dissection dishes
Living Systems Instrumentation
DD-50-S or DD-90-S
52 mm diameter x 18 mm H (Sylgard Depth ~6mm) or 93 mm x 22 mm
Jeffery, F., La Mattina, C., Tuton-Blasingame, T., Hsia, Y., Gnesa, E., Zhao, L., Franz, A., Vierra, C. Microdissection of Black Widow Spider Silk-producing Glands. J. Vis. Exp. (47), e2382, doi:10.3791/2382 (2011).