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| + | <div tabindex="0" class="onclick-menu"> |
| + | <ul class="onclick-menu-content" style="list-style:none;"> |
| + | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton">home</a></li> |
| + | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Team">team</a></li> |
| + | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Project">project</a></li> |
| + | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Human">human practices</a></li> |
| + | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Notebook">notebook</a></li> |
| + | </ul> |
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− | <center><img src="http://upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE_Electrophoresis.png" width="750px"></center> | + | <center><img src="https://static.igem.org/mediawiki/2015/b/bb/NAIT_SDSPAGE.png" width="750px"></center> |
| + | <p style="font-size:10px">Source: http://upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE_Electrophoresis.png</p> |
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− | <center><img src="http://www.bio-rad.com/webroot/web/images/lsr/products/electrophoresis/product_overlay_content/global/lsr_biosafe_coomasie_gel.jpg"></center> | + | <center><img src="https://static.igem.org/mediawiki/2015/b/b0/Lsr_biosafe_coomasie_gel.jpg"></center> |
| + | <p><font size="1px">[1] Bio-rad.com, 'Coomassie Stains', 2015. [Online]. Available: http://www.bio-rad.com/webroot/web/images/lsr/products/electrophoresis/product_overlay_content/global/lsr_biosafe_coomasie_gel.jpg. [Accessed: 14- Jun- 2015].</font></p> |
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| <a class="accordion-section-title" href="#accordion-2" style="background-color:#FBB252">The Problem</a> | | <a class="accordion-section-title" href="#accordion-2" style="background-color:#FBB252">The Problem</a> |
| <div id="accordion-2" class="accordion-section-content"> | | <div id="accordion-2" class="accordion-section-content"> |
− | <p>Difficulties with silver staining arise when the molecular weight markers are re- | + | <p>However, there are limitations to using the silver staining technique. The primary issue with the technique is that the users lose the ability to use the color coded molecular weight marker as a reference post-staining. To combat this issue, researchers poke holes into the polyacrylamide gel so that they can retain their molecular weight ladder reference points. However, by doing this we ruin the integrity of the gel making the staining process much more likely to damage or destroy the fragile gel. </p> |
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− | colored golden-brown in the staining process. Markers offer evenly distributed proteins
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− | that show bands of equal intensity and known size. Researchers can compare these
| + | <center><img src="https://static.igem.org/mediawiki/2015/a/a9/NAIT_SilverStain.jpeg" width="750px"> |
| + | <p style="font-size:10px">Source:http://labs.mmg.pitt.edu/gjoerup/silver_stain.jpg</p> |
| + | </center><br> |
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− | bands with their sample and identify the protein they are looking for based on its size. A
| + | <p>Researchers who have used the technique in the past have also expressed concerns on the time it takes to develop the gel and stain; the possibility of “over-staining” the gel, and the generation of false positives due to the sensitivity of the technique. (An example would be the solutions reacting with particles in the air to provide an unneeded background on the gel) </p> |
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− | subset of these markers has color-coded standard proteins to facilitate the identification
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− | of each band. Post-silver staining, the users lose the ability to use the color code as a | + | |
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− | reference.</p>
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− | <center><img src="http://labs.mmg.pitt.edu/gjoerup/silver_stain.jpg" width="750px"></center>
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− | <a class="accordion-section-title" href="#accordion-3">Our Goal</a> | + | <a class="accordion-section-title" href="#accordion-3" style="background-color: #f7e133">Our Goal and Solution</a> |
| <div id="accordion-3" class="accordion-section-content"> | | <div id="accordion-3" class="accordion-section-content"> |
− | <p>Our goal is to develop a marker that, when interacting with the reagents used in | + | <p>Our goal is to develop a marker that, when interacting with the reagents used in the staining protocol, will stain in colour and in specific positions. This technology will aid in the identification of the protein(s) of interest post-staining. In order to do so, we will investigate how specific amino acids react with silver staining reagents. After determining specific motifs, we aim to create novel proteins that contain an excess of that motif, particular amino acid and/or chemical modifications that will generate a specific color after treating it with silver staining reagents. To obtain such proteins, we will introduce novel nucleotide sequences into a plasmid by in vitro transcription translation. <i>E. coli</i> cells with expression vectors will then be transformed with the new plasmids.</p> <br> |
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− | the staining protocol, will develop colour bands in specific positions so as to help in the | + | <p>By testing different techniques to develop and stain the gels we hope to see if the expressed concerns are still an issue, namely time taken to develop the gels and stain them effectively. To investigate this we will use BioRad’s recommended protocol, our protocol developed in-house, and a technique documented in <i>Electrophoresis.</i></p> |
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− | identification of the protein(s) of interest post-staining. In order to do so, investigation of
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− | how specific amino acids react with silver staining reagents is underway by our team.
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− | This will have as an outcome the creation of novel proteins that contain an excess of a
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− | particular amino acid and/or chemical modifications that will generate a specific colour
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− | after treating it with silver staining reagents. To obtain such proteins, the introduction of
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− | novel nucleotide sequences into a plasmid would be done first by in vitro transcription
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− | translation and later by transforming E. coli cells with expression vectors.</p>
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− | </div><!--end .accordion-section-content-->
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− | </div><!--end .accordion-section-->
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− | <div class="accordion-section">
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− | <a class="accordion-section-title" href="#accordion-4">Our Solution</a>
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− | <div id="accordion-4" class="accordion-section-content">
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− | <p>Design novel protein sequences that will stain in colour.</p>
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| </div><!--end .accordion-section-content--> | | </div><!--end .accordion-section-content--> |
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| <div class="accordion-section"> | | <div class="accordion-section"> |
− | <a class="accordion-section-title" href="#accordion-5">Why Does this Matter?</a> | + | <a class="accordion-section-title" href="#accordion-5" style="background-color:#afcf64">Why Does this Matter?</a> |
| <div id="accordion-5" class="accordion-section-content"> | | <div id="accordion-5" class="accordion-section-content"> |
| <p>With the current technique used today, researchers poke holes into the PA gel so that they can retain their molecular weight ladder reference points. Not only does this take time to do, but it also ruins the integrity of the gel making the staining process much more likely to damage the fragile gel.</p> | | <p>With the current technique used today, researchers poke holes into the PA gel so that they can retain their molecular weight ladder reference points. Not only does this take time to do, but it also ruins the integrity of the gel making the staining process much more likely to damage the fragile gel.</p> |