کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10534096 | 961904 | 2005 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Optical zymography for specific detection of urokinase plasminogen activator activity in biological samples
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موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
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چکیده انگلیسی
Zymography techniques are routinely used to quantify proteolytic activity. In the current study, we describe an optical zymographic procedure that specifically detects urokinase-type plasminogen activator (uPA) activity in biological samples. The method employs a synthetic polymeric uPA fluorescent probe, which is copolymerized in sodium dodecyl sulfate (SDS)-polyacrylamide gel. Following electrophoresis and renaturation, enzymatic digestions of the substrate in 50Â mM of Tris buffer at pH 7.4 generates fluorescence emission at 695Â nm. The enzymatic activities can be analyzed directly by conventional gel imaging systems with a detection limit of 40Â pg. This protocol is fast (hours) and does not require staining and destaining steps. The procedure is independent of plasminogen and, therefore, can efficiently distinguish the active two-chain uPA from its proenzyme. Densitometry analysis demonstrated a highly correlative relationship (r2Â =Â 0.999) between the amount of uPA (over the range of 0.1-8.0Â ng) and the average intensity of the fluorescent band. We were able to directly measure uPA activities in different cancer cell lines. This newly developed technique could be expanded to nearly all proteases, including the ones that cannot be analyzed by traditional zymography.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 338, Issue 1, 1 March 2005, Pages 151-158
Journal: Analytical Biochemistry - Volume 338, Issue 1, 1 March 2005, Pages 151-158
نویسندگان
Benedict Law, Jong-Kai Hsiao, Thomas H. Bugge, Ralph Weissleder, Ching-Hsuan Tung,