Biology Reference
In-Depth Information
that remain unknown) proteins that lack signal sequences, such as glycolytic
proteins (3,4,5) .
The relatively small number of culture supernatant proteins (CSPs),
compared with proteins in the cytoplasm, simplifies proteomic analysis.
However, several aspects can make two-dimensional gel electrophoresis (2-DE)
of CSPs challenging (6) . These include (1) the presence of basic CSPs, which
can become insoluble during isoelectric focusing, (2) the presence of hyaluronic
acid, which can interfere with isoelectric focusing, (3) the presence of peptides
in complex media, such as Todd-Hewitt broth, and (4) the abundance of specific
exoproteins such as the cysteine protease SpeB, which can mask less abundant
proteins (7) .
Analysis typically involves three steps. First, exoproteins are isolated from
bacterial cultures. The aim of this process is to retain as many proteins as
possible while eliminating components that inhibit isoelectric focusing. To do
so, proteins are enzymatically treated and precipitated to remove salts and
other non-protein components. Second, the proteins are resolved by one- or
two-dimensional gel electrophoresis ( see Fig. 1 ). Third, proteins are visualized
and the composition and quantities of protein spots determined. The protocol
described below uses a mini-gel format that is relatively fast and requires small
culture volumes compared with large-format two-dimensional gel electropho-
resis.
Mr
116
45
31
21
4
7
pI
Fig. 1. Two-dimensional gel electrophoresis of culture supernatant proteins. Proteins
were isolated from wild-type Streptococcus pyogenes strain NZ131 after 18 h of
culture in 40 mL of chemically defined medium. Hundred micrograms of protein was
separated with a 7-cm immobilized gradient strip (pH 4-7) (IPG; GE Biosciences) and
SDS-PAGE. Proteins were stained with SYPRO Ruby.
 
Search WWH ::




Custom Search