کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8420207 | 1545772 | 2008 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Single-step immunoaffinity purification and functional reconstitution of human phagocyte flavocytochrome b
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کلمات کلیدی
DMFmAbCMCCNBrHEPESPMSFDOPADTTNADPHMonoclonal antibody - آنتی بادی مونوکلونالEDTA - اتیلن دی آمین تترا استیک اسید Ethylenediaminetetraacetic acid - اتیلینیدامین تتراستیک اسیدphosphatidic acid - اسید فسفاتیدیکSDS-PAGE - الکتروفورز ژل پلی آکریل آمیدsodium dodecylsulfate polyacrylamide gel electrophoresis - الکتروفورز ژل پلی اکریللید سدیم دودسیل سولفاتoctylglucoside - اکتیل گلوکوزیدReconstitution - بازسازیcyanogen bromide - برومید سایانوژنdimethylformamide - دی متیل فرمالیدdithiothreitol - دیتیوتریتولcyt b - سیت بcritical micelle concentration - غلظت متیل مهمphosphatidylcholine - فسفاتیدیل کولینphenylmethylsulfonyl fluoride - فنیل متیل سولفونیل فلورایدneutrophil - نوتروفیلnicotinamide adenine dinucleotide - نیکوتین آمید adenine dinucleotidePurification - پاکسازی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیوتکنولوژی یا زیستفناوری
پیش نمایش صفحه اول مقاله
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چکیده انگلیسی
Human neutrophil flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that generates high levels of superoxide in the multisubunit NADPH oxidase complex. Since Cyt b is currently isolated in limited quantities, improved methods for purification from low levels of starting membranes (from both neutrophils and other expressing cell types) are important for the analysis of structure and catalytic mechanism. In the present study, the epitope-mapped monoclonal antibody CS9 was coupled to Sepharose beads and used as an affinity matrix for single-step immunoaffinity purification of Cyt b. Following solubilization of both human neutrophil and PLB-985 membrane fractions in the nonionic detergent octylglucoside, Cyt b was absorbed on the CS9-Sepharose affinity matrix and purified protein was eluted under non-denaturing conditions with an epitope-mimicking peptide. The high efficiency of this isolation procedure allowed Cyt b to be reproducibly purified from readily obtainable levels of starting membrane fractions (9Â ÃÂ 108 cell equivalents of neutrophil membranes and 2Â ÃÂ 109 cell equivalents of PLB-985 membranes). Since Cyt b could be affinity-purified in the detergent octylglucoside, high-level functional reconstitution was carried out directly on elution fractions by simple addition of solubilized phospholipid and subsequent dialysis for detergent removal. To our knowledge, this study describes the most efficient method for generating purified, functionally-reconstituted Cyt b and should facilitate analyses that require a highly-defined NADPH oxidase system.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 329, Issues 1â2, 1 January 2008, Pages 201-207
Journal: Journal of Immunological Methods - Volume 329, Issues 1â2, 1 January 2008, Pages 201-207
نویسندگان
Connie I. Lord, Marcia H. Riesselman, Jeannie M. Gripentrog, James B. Burritt, Algirdas J. Jesaitis, Ross M. Taylor,