Difference between revisions of "Team:NAIT Edmonton/Trial11"

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Team NAIT 2015

The Project

Sodium Dodecyl Sulphide Polyacrylamide Gel Electrophoresis (SDS PAGE) is one of the most prominent techniques used in protein research. However, one of its limitations, more specifically the loss of an accurate point of reference post staining, is attributed to inefficiency and the wasting of time during the research process. From past observations, some proteins inherently produce certain colours post-silver staining. We hypothesized that certain configurations of amino acids produces specific colours when reacting with the reagents of silver staining. In the hopes of creating a new molecular weight ladder, our team designed sequences to code for novel proteins that produce colour. Coloured proteins can also pave the way for new types of colorimetric assays and a less expensive and more accessible counterpart to antibody tags in the future.

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.

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