کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10756397 | 1050383 | 2014 | 5 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Combinatory use of cell-free protein expression, limited proteolysis and mass spectrometry for the high-throughput protein domain identification
ترجمه فارسی عنوان
استفاده ترکیبی از بیان پروتئین سلولی، پروتئولیز محدود و طیف سنجی جرم برای شناسایی دامنه پروتئین با پروتئین بالا
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کلمات کلیدی
با توان بالا بیان پروتئین بدون سلول، پروتئین متصل به دانه پروتئولیت محدود، طیف سنجی جرمی، دامنه،
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
چکیده انگلیسی
The structural domains of proteins have often been identified through the use of limited proteolysis. In structural genomics studies, it is necessary to carry this out in a high-throughput manner. Here, we constructed a novel high-throughput system, which consists of cell-free protein expression and one-step affinity purification, followed by limited proteolysis using a unique new method, referred to “on beads method”. All these steps were carried out on 96-well plate formats and completed in two days, even by manual handling. The merits of the new method versus the conventional one are as follows: (1) experimental times are reduced, (2) the sample preparation for limited proteolysis experiments is simplified, and (3) both protein purification and limited digestion can be performed “in situ” on the same sample plate. This preparation method is therefore suitable for highly automated, proteolytic analyses coupled to mass spectrometry techniques at a micro-scale protein expression level. The resulting protease-resistant fragments were analyzed by MALDI-TOF-MS and protein domains of 34 mouse cDNA products were identified with this system.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochemical and Biophysical Research Communications - Volume 444, Issue 4, 21 February 2014, Pages 480-484
Journal: Biochemical and Biophysical Research Communications - Volume 444, Issue 4, 21 February 2014, Pages 480-484
نویسندگان
Xinchun Shen, Siyuan Chen, Heng Ge,