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Technical Note

Pre-extraction Sample Handling by Automated Frozen Disruption Significantly Improves Subsequent Proteomic Analyses

R. Hussain Butt^† and Jens R. Coorssen*^†^‡^§

Departments of Physiology & Biophysics, Biochemistry & Molecular Biology, and Cell Biology and Anatomy, and Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, AB, Canada

J. Proteome Res., 2006, 5 (2), pp 437–448

DOI: 10.1021/pr0503634

Publication Date (Web): December 22, 2005

†

Departments of Physiology & Biophysics.

To whom correspondence should be addressed. Room 174 Heritage Medical Research Building, 3330 Hospital Drive NW, Calgary AB Canada, T2N 4N1. Tel: (403) 220-2422. Fax. (403) 283-7137. E-mail: jcoorsse@ucalgary.ca.

‡

Biochemistry & Molecular Biology.

Cell Biology and Anatomy.

Abstract

Here we quantitatively characterize two common homogenization strategies in the analysis of tissue proteomes: classical manual homogenization (MH) and an automated frozen disruption (AFD) technique. In a variety of tissues, many proteins were more efficiently extracted, resolved and detected, with high reproducibility after AFD, amounting to as much as 2% of the total resolved proteome. The benefits of AFD over MH are 2-fold: (1) AFD yields a much more thorough homogenate than MH; and (2) as a deep frozen alternative, AFD maintains a level of biological complexity that is not retained during MH. Thus, AFD coupled with refined 2DE protocols and Sypro Ruby staining yields quantitative proteomic analyses.

Keywords: proteomics • membrane proteins • tissue homogenization • protein extraction

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