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An Economical High-Throughput Protocol for Multidimensional Fractionation of Proteins
Joint Authors
Gopala Krishna, Varun
Layfield, Robert
Tooth, David John
Source
International Journal of Proteomics
Issue
Vol. 2012, Issue 2012 (31 Dec. 2012), pp.1-10, 10 p.
Publisher
Hindawi Publishing Corporation
Publication Date
2012-09-12
Country of Publication
Egypt
No. of Pages
10
Main Subjects
Natural & Life Sciences (Multidisciplinary)
Biology
Abstract 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.
American Psychological Association (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
Modern Language Association (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
American Medical Association (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
Data Type
Journal Articles
Language
English
Notes
Includes bibliographical references
Record ID
BIM-494460