Biology Reference
In-Depth Information
3. Methods
Samples stored in liquid nitrogen can be used, but snap-freezing
causes cell rupture and may also damage the conformation of the ECM.
Thus, it is preferable to process samples immediately without freezing.
The protein subfractionation methodology is summarized in Fig. 1 .
3.1. Sample
Preparation
Typically, 50 mg of tissue is suffi cient to obtain an adequate amount
of proteins in each extraction step for proteomics analysis. Prior to
extraction, tissue samples must be weighed and diced into smaller
Fig. 1. Biochemical subfractionation of the cardiac proteome: ( a ) schematic representation of the biochemical subfraction-
ation for improved proteomics analysis of cardiovascular tissues (adapted from: Didangelos et al. ( 9 ) ), ( b ) a representative
Coomassie blue-stained 1-D gel comparing the NaCl, SDS, and guanidine extracts from different murine cardiac samples.
Note that the SDS extracts are the most complex and are subjected to further analysis by 2-DE (see Fig. 2 ) .
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