Bio-Rad ReadyPrep Sequential Extraction Kit - Manual (Page 13)

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Two-dimensional gel analysis of extracts from E. coli.
E. coli W3110 was collected by centrifugation. The cell pellet was
suspended in Reagent 1 and the cells were lysed by sonication. One
portion of the sonicated cell suspension, containing 200 µg of protein,
was diluted in extraction solution 3. The proteins soluble in solution 3
were separated by 2-D PAGE as a “whole cell extract” (A). Another
portion of the sonicated cell suspension was subjected to the
sequential extraction protocol. (B) 2-D PAGE separation of 200 µg of
protein from Extract 1. (C) 2-D PAGE separation of 200 µg of protein
from Extract 2. (D) 2-D PAGE separation of 200 µg of protein from
Extract 3. First-dimension separation was by isoelectric focusing
using ReadyStrip IPG strips pH 4 to pH 7. The second-dimension
separation was by SDS-PAGE in an 8%T–16%T polyacrylamide
gradient gel.
A B
C D
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