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

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<center><div class="top_slogan">The Project</div></center>
 
    
 
    
 
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<h2>Column 1</h2>
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<h1>Background</h1>
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<p><a href="/">456 Berea Street Home</a></p>
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<p><a href="/lab/developing_with_web_standards/csslayout/2-col/">Simple 2 column CSS layout</a></p>
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<p>The structural and functional study of the proteins expressed by a genome is
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<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Mauris vel magna. Mauris risus nunc, tristique varius, gravida in, lacinia vel, elit. Nam ornare, felis non faucibus molestie, nulla augue adipiscing mauris, a nonummy diam ligula ut risus. Praesent varius. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus.</p>
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<p>Nulla a lacus. Nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Fusce pulvinar lobortis purus. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec semper ipsum et urna. Ut consequat neque vitae felis. Suspendisse dapibus, magna quis pulvinar laoreet, dolor neque lacinia arcu, et luctus mi erat vestibulum sem. Mauris faucibus iaculis lacus. Aliquam nec ante in quam sollicitudin congue. Quisque congue egestas elit. Quisque viverra. Donec feugiat elementum est. Etiam vel lorem.</p>
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called proteomics. This relatively novel science uses different methodologies in order to
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separate and identify specific proteins of interest. Among these techniques, SDS-PAGE
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plays an essential role due to its high sensitivity, low sample volume requirement, and
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high popularity. Negatively charged proteins migrate towards the positive electrode
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according to their size and charge. Smaller proteins migrate further in a given amount of
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time. As proteins are separated in this manner, users load molecular weight standards
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to estimate the size (in kDa) of the proteins present in their sample. Once the proteins of
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a single sample have been isolated and are embedded in the polyacrylamide (PA) gel
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matrix, staining procedures are used to visualize them.</p>
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<br>
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<p>Organic dyes, such as Coomassie blue, can be used for this purpose;
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 +
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
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 +
protocol, thus making it one of the most commonly used staining methods. </p>
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<br><br>
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<h1>The Problem </h1>
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<p>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.</p>
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<br><br>
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<h1>Our Goal</h1>
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<p>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.</p>
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 +
<br>
 
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       <div id="nav_bar">
 
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Revision as of 22:49, 9 June 2015

Team NAIT 2015
The Project

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.