An Economical High-Throughput Protocol for Multidimensional Fractionation of Proteins
المؤلفون المشاركون
Gopala Krishna, Varun
Layfield, Robert
Tooth, David John
المصدر
International Journal of Proteomics
العدد
المجلد 2012، العدد 2012 (31 ديسمبر/كانون الأول 2012)، ص ص. 1-10، 10ص.
الناشر
Hindawi Publishing Corporation
تاريخ النشر
2012-09-12
دولة النشر
مصر
عدد الصفحات
10
التخصصات الرئيسية
العلوم الطبيعية والحياتية (متداخلة التخصصات)
الأحياء
الملخص EN
A sequential protocol of multidimensional fractionation was optimised to enable the comparative profiling of fractions of proteomes from cultured human cells.
Differential detergent fractionation was employed as a first step to obtain fractions enriched for cytosolic, membrane/organelle, nuclear, and cytoskeletal proteins.
Following buffer exchange using gel-permeation chromatography, cytosolic proteins were further fractionated by 2-dimensional chromatography employing anion-exchange followed by reversed-phase steps.
Chromatographic fractions were shown to be readily compatible with 1- and 2-dimensional gel electrophoresis or with direct analysis by mass spectrometry using linear-MALDI-TOF-MS.
Precision of extraction was confirmed by reproducible SDS-PAGE profiles, MALDI-TOF-MS spectra, and quantitation of trypsinolytic peptides using LC-MS/MS (MRM) analyses.
Solid phases were immobilised in disposable cartridges and mobile-phase flow was achieved using a combination of centrifugation and vacuum pumping.
These approaches yielded parallel sample handling which was limited only by the capacities of the employed devices and which enabled both high-throughput and experimentally precise procedures, as demonstrated by the processing of experimental replicates.
Protocols were employed at 10 mg scale of extracted cell protein, but these approaches would be directly applicable to both smaller and larger quantities merely by adjusting the employed solid- and mobile-phase volumes.
Additional potential applications of the fractionation protocol are briefly described.
نمط استشهاد جمعية علماء النفس الأمريكية (APA)
Tooth, David John& Gopala Krishna, Varun& Layfield, Robert. 2012. An Economical High-Throughput Protocol for Multidimensional Fractionation of Proteins. International Journal of Proteomics،Vol. 2012, no. 2012, pp.1-10.
https://search.emarefa.net/detail/BIM-494460
نمط استشهاد الجمعية الأمريكية للغات الحديثة (MLA)
Tooth, David John…[et al.]. An Economical High-Throughput Protocol for Multidimensional Fractionation of Proteins. International Journal of Proteomics No. 2012 (2012), pp.1-10.
https://search.emarefa.net/detail/BIM-494460
نمط استشهاد الجمعية الطبية الأمريكية (AMA)
Tooth, David John& Gopala Krishna, Varun& Layfield, Robert. An Economical High-Throughput Protocol for Multidimensional Fractionation of Proteins. International Journal of Proteomics. 2012. Vol. 2012, no. 2012, pp.1-10.
https://search.emarefa.net/detail/BIM-494460
نوع البيانات
مقالات
لغة النص
الإنجليزية
الملاحظات
Includes bibliographical references
رقم السجل
BIM-494460
قاعدة معامل التأثير والاستشهادات المرجعية العربي "ارسيف Arcif"
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