کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5504553 | 1536762 | 2017 | 23 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat
ترجمه فارسی عنوان
استراتژی ساده ی حذف برای پروتئین های بیوتینیل شده که به دانه های کنجوبت استرپتاویدین متصل می شوند با استفاده از بیوتین بیش از حد و گرما
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
چکیده انگلیسی
Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to traditional co-immunoprecipitation. In this protocol, a mutant biotin ligase promiscuously labels proximal binding partners with biotin, and resulting biotinylated proteins are purified using streptavidin conjugated beads. This approach does not require preservation of protein complexes in vitro, making it an ideal approach to identify transient or weak protein complexes. However, due to the high affinity bond between streptavidin and biotin, elution of biotinylated proteins from streptavidin conjugated beads requires harsh denaturing conditions, which are often incompatible with downstream processing. To effectively release biotinylated proteins bound to streptavidin conjugated beads, we designed a series of experiments to determine optimal binding and elution conditions. Interestingly, the concentrations of SDS and IGEPAL-CA630 during the incubation with streptavidin conjugated beads were the key to effective elution of biotinylated proteins using excess biotin and heating. This protocol provides an alternative method to isolate biotinylated proteins from streptavidin conjugated beads that is suitable for further downstream analysis.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochemical and Biophysical Research Communications - Volume 493, Issue 4, 2 December 2017, Pages 1522-1527
Journal: Biochemical and Biophysical Research Communications - Volume 493, Issue 4, 2 December 2017, Pages 1522-1527
نویسندگان
Joleen S. Cheah, Soichiro Yamada,