Garb Jessica E
Department of Biological Sciences, University of Massachusetts Lowell;
J Vis Exp. 2014 Nov 3(93):e51618. doi: 10.3791/51618.
Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.
毒液是化学组成复杂的分泌物，通常包含众多具有不同生理活性的蛋白质和肽。毒液蛋白的功能表征具有重要的生物医学应用，包括鉴定药物先导物或细胞受体探针。蜘蛛是有毒生物中物种最为丰富的类群，但只有少数物种的毒液得到了充分了解，部分原因是从小动物身上采集微量毒液存在困难。本文介绍了一种使用电刺激从蜘蛛采集毒液的方法，并以西黑寡妇（间斑寇蛛）为例展示了该过程。所采集的毒液可用于多种下游分析，包括通过质谱直接鉴定蛋白质、功能测定以及刺激毒液基因表达以进行转录组学研究。与从整个腺体匀浆中分离毒液的方法相比，该技术具有优势，后者无法将真正的毒液成分与不作为毒液一部分分泌的细胞蛋白区分开来。代表性结果表明，使用质谱法可从采集的样本中检测到已知的毒液肽。毒液采集过程之后是蜘蛛毒腺解剖方法，结果表明这能在序列水平上对毒液表达的蛋白质和肽进行表征。
