Background
The structural and functional study of the proteins expressed by a genome is
called proteomics. This relatively novel science uses different methodologies in order to
separate and identify specific proteins of interest. Among these techniques, SDS-PAGE
plays an essential role due to its high sensitivity, low sample volume requirement, and
high popularity. Negatively charged proteins migrate towards the positive electrode
according to their size and charge. Smaller proteins migrate further in a given amount of
time. As proteins are separated in this manner, users load molecular weight standards
to estimate the size (in kDa) of the proteins present in their sample. Once the proteins of
a single sample have been isolated and are embedded in the polyacrylamide (PA) gel
matrix, staining procedures are used to visualize them.
Organic dyes, such as Coomassie blue, can be used for this purpose;
nevertheless, their low sensitivity and a detection range that goes from 1 to 50 ng can
be a challenge for detecting low abundance proteins (Jin, Huang, Yoo, & Choi, 2006). A
higher sensitivity can be achieved by fluorescent staining techniques (from 0.1 to 10
ng.); however, UV instruments are necessary in order to read the data (Jin et al., 2006).
The most sensitive method up to date is radiolabeling, but the requirement of hazardous
isotopes and their complex management makes it a complicated procedure (Jin et al.,
2006). Silver staining is a method that offers great sensitivity and an easy to handle
protocol, thus making it one of the most commonly used staining methods.
The Problem
Difficulties with silver staining arise when the molecular weight markers are re-
colored golden-brown in the staining process. Markers offer evenly distributed proteins
that show bands of equal intensity and known size. Researchers can compare these
bands with their sample and identify the protein they are looking for based on its size. A
subset of these markers has color-coded standard proteins to facilitate the identification
of each band. Post-silver staining, the users lose the ability to use the color code as a
reference.
Our Goal
Our goal is to develop a marker that, when interacting with the reagents used in
the staining protocol, will develop colour bands in specific positions so as to help in the
identification of the protein(s) of interest post-staining. In order to do so, investigation of
how specific amino acids react with silver staining reagents is underway by our team.
This will have as an outcome the creation of novel proteins that contain an excess of a
particular amino acid and/or chemical modifications that will generate a specific colour
after treating it with silver staining reagents. To obtain such proteins, the introduction of
novel nucleotide sequences into a plasmid would be done first by in vitro transcription
translation and later by transforming E. coli cells with expression vectors.
