Bacterial Pathogenesis

Spot Identification
appropriate image analysis software. While purpose-built laser densito-
meters are still used extensively to analyze silver-stained gels, modem
desk-top document scanners usually give comparable results when fitted
with an adaptor to allow scanning of transparencies. Charge-coupled
device (CCD) cameras have also been used for this purpose. If gels are
stained with a fluorescent dye the most practical option is to use a fluor-
escence imager. However, it is important to ensure that the system is com-
patible with the fluorescent stain employed - not all of them produce light
of the correct excitation wavelength or can sensitively detect light at the
relevant emission Wavelength.
Since gels of bacterial lysates may contain over 2000 protein spots,
studies to investigate patterns of protein expression are greatly facilitated
by use of a gel analysis software package, which will automatically detect
spots, measure them, and match them between gels. Several sophisticated
packages are available including MELANIE 11 and PDQUEST, which are
UNIX-based and designed to run on dedicated workstation computers,
and PHORETIX 2D, which is a versatile Windows-based package that
runs on PCs. Once data are retrieved from gels the software can be used
to generate 2D protein databases to allow searches for changes in patterns
of protein expression. Recently, some of these databases have become
accessible via the Internet (e.g. the Swiss 2D database at Internet location
http:llexpasy.hcuge.ch/ch2dlch2d-top.html). The main advantage of
this development is that it provides an easy link with other relevant data-
bases. For instance, a protein that is highly induced under a particular set
of conditions can be identified by calling up an image of a reference gel
via the Internet and 'clicking' on the corresponding spot with a mouse.
Any information available for the spot of interest is then automatically
displayed and if the identity of the protein is known, a link to a sequence
database such as SWISSPROT will be provided.
Protein microsequencing
Frequently the end-point of 2D-GE is identification of a polypeptide of
interest to determine whether it is known or novel. Either protein
microsequencing or mass spectrometry (MS) can be used for this purpose,
and the strategy employed is usually dictated by the amount of material
that can be obtained for analysis and the equipment available. An addi-
tional consideration is that sequencing provides a route for the cloning of
the genes encoding proteins of interest, through the use of synthetic
oligonucleotide probes and library screening or the polymerase chain
reaction (PCR). While it is possible to obtain de novo sequence data by MS,
it is technically more difficult. Whichever approach is used, the fewer
steps involved in handling the sample the better as each results in loss of
material and hence reduces sensitivity.
In theory, for microbes that have less than 8000 proteins and completely-
sequenced genomes, a contiguous N-terminal sequence of three amino
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