كتاب
Calpain zymography with casein or fluorescein isothiocyanate casein.
| العنوان: | Calpain zymography with casein or fluorescein isothiocyanate casein. |
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| المؤلفون: | Arthur, J. Simon C., Mykles, Donald L. |
| المساهمون: | Elce, John S. |
| المصدر: | Arthur , J S C & Mykles , D L 2000 , Calpain zymography with casein or fluorescein isothiocyanate casein. in J S Elce (ed.) , Calpain methods and protocols . vol. 144 , Methods in Molecular Biology (Clifton, N.J.) , vol. 144 , Springer , pp. 109-116 . https://doi.org/10.1385/1-59259-050-0:109Test |
| بيانات النشر: | Springer |
| سنة النشر: | 2000 |
| المجموعة: | Discovery - University of Dundee Online Publications |
| مصطلحات موضوعية: | /dk/atira/pure/subjectarea/asjc/1300/1312, name=Molecular Biology, /dk/atira/pure/subjectarea/asjc/1300/1311, name=Genetics |
| الوصف: | Methods for analyzing enzyme activity after electrophoresis in acrylamide gels have been described for a variety of proteins, including several proteases and kinases. The application of this approach to detect calpain activity by casein zymography was first described by Raser et al. in 1995 (1), and has proved itself by its successful application in several laboratories interested in calpain. Briefly, samples are run under nondenaturing conditions in the presence of EDTA on acrylamide gels in which casein has been copolymerized. After electrophoresis the gels are incubated overnight in buffer containing Ca2+ and reducing agent to activate calpain. During this time, casein in the region of an active calpain band is digested to fragments sufficiently small to diffuse out of the gel, and the calpain itself is also extensively degraded. The gel is then stained conventionally in Coomassie brilliant blue, giving rise to a clear band in the dark blue gel. These gels are not very easy to photograph, and a useful modification is to use fluorescein isothiocyanate (FITC)-casein in place of casein in the gels. The FITC-casein system is also more sensitive. In this case, the gels are viewed and photographed with ultraviolet (UV) illumination, and proteinase activity produces dark (nonfluorescent) zones in a bright background. The zymogram method as applied to calpain is still undergoing modifications in many laboratories, as shown for example in Chapter 14, in which some details of the method differ. |
| نوع الوثيقة: | book part |
| اللغة: | English |
| ردمك: | 978-1-61737-105-9 1-61737-105-X |
| العلاقة: | https://discovery.dundee.ac.uk/en/publications/588a73ed-58ed-403c-8360-8444dfeead11Test; urn:ISBN:978-1-61737-105-9 |
| DOI: | 10.1385/1-59259-050-0:109 |
| الإتاحة: | https://doi.org/10.1385/1-59259-050-0:109Test https://discovery.dundee.ac.uk/en/publications/588a73ed-58ed-403c-8360-8444dfeead11Test http://www.scopus.com/inward/record.url?scp=0033650175&partnerID=8YFLogxKTest |
| حقوق: | info:eu-repo/semantics/restrictedAccess |
| رقم الانضمام: | edsbas.AECD4D09 |
| قاعدة البيانات: | BASE |
| ردمك: | 9781617371059 161737105X |
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| DOI: | 10.1385/1-59259-050-0:109 |