Team:Lethbridge Canada/results

From 2013hs.igem.org

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<p>Below is a Slot Blot done to confirm the presence of a Oxytocin-Neurophysin. During the synthesis of our gene, histidine tags were added following the protein. A Slot Blot test works by utilizing two types of antibodies, and will fluoresce if our construct is present. The primary antibody is sensitive to histidine and accordingly, will bind to it. The second anitbody is sensitive to the first, but has a florescent protein attached to another arm. Should the secondary anitbody bind to the primary antibody, they will both remain attached to the histidine tag on the protein and when when induced, will glow. Accordingly, we can detect this chemiluminescence and confirm the presence of our protein.</p>
<p>Below is a Slot Blot done to confirm the presence of a Oxytocin-Neurophysin. During the synthesis of our gene, histidine tags were added following the protein. A Slot Blot test works by utilizing two types of antibodies, and will fluoresce if our construct is present. The primary antibody is sensitive to histidine and accordingly, will bind to it. The second anitbody is sensitive to the first, but has a florescent protein attached to another arm. Should the secondary anitbody bind to the primary antibody, they will both remain attached to the histidine tag on the protein and when when induced, will glow. Accordingly, we can detect this chemiluminescence and confirm the presence of our protein.</p>
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<th>Lane #</th>
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<th>Contents</th>
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<td>Lane 1:</td>
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<td>100&mu;L Oxytocin in 200&mu;L TBS</td>
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<td>Lane 2:</td>
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<td>: 200&mu;L Oxytocin in 100&mu;L TBS</td>
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<td>Lane 3:</td>
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<td>250 &mu;L Oxytocin in 50 &mu;L TBS</td>
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<td>Lane 4:</td>
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<td>300 &mu;LOxytocin</td>
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<td>Lane 5:</td>
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<td>25pM TruB-His positive control</td>
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<img src="https://static.igem.org/mediawiki/2013hs/a/af/Lethhs_2013_oxt_histag.png" alt="Slot Blot confirming His-Tag" />
<img src="https://static.igem.org/mediawiki/2013hs/a/af/Lethhs_2013_oxt_histag.png" alt="Slot Blot confirming His-Tag" />
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Revision as of 23:38, 21 June 2013



Silver Stained Tris-Tricine PAGE

Lane Contents Volume
1 Protein Molecular Weight Marker 20μL
2 Cbf5-Nop10-Gar1 20μL
3 Buffer A wash 30μL
4 Buffer B wash 30μL
5 Buffer E Wash 30μL
6 S-30 s/N 30μL
7 Buffer E – Concentrated Preprooxyphysin 30μL
Gel Gel

To detect our prepro-oxyphysin proteins we did a Silver Stain 16.5% Tris-Tricine PAGE. The reason for doing a Silver Stain is that it is 10x more sensitive than the standard Coomassie stain. The tricine in the Tris-TRicine PAGE allows for better and more distinct separation of small proteins in the gel. In Lane 2, we did a control of Cbf5-Nop10-Gar1 proteins. Cbf5 is 43 kDa, Gar1 is 12.3 kDa and Nop10 is only 7.2 kDa. Nop 10 is outlined in red. Prepro-oxyphysin is slightly larger than Nop10 at 10kDa. The bands we suspect are prepro-oxyphysin are outlined by orange.

Anti-His Slot Blot

Below is a Slot Blot done to confirm the presence of a Oxytocin-Neurophysin. During the synthesis of our gene, histidine tags were added following the protein. A Slot Blot test works by utilizing two types of antibodies, and will fluoresce if our construct is present. The primary antibody is sensitive to histidine and accordingly, will bind to it. The second anitbody is sensitive to the first, but has a florescent protein attached to another arm. Should the secondary anitbody bind to the primary antibody, they will both remain attached to the histidine tag on the protein and when when induced, will glow. Accordingly, we can detect this chemiluminescence and confirm the presence of our protein.

Lane # Contents
Lane 1: 100μL Oxytocin in 200μL TBS
Lane 2: : 200μL Oxytocin in 100μL TBS
Lane 3: 250 μL Oxytocin in 50 μL TBS
Lane 4: 300 μLOxytocin
Lane 5: 25pM TruB-His positive control
Slot Blot confirming His-Tag

1%Agarose Gel of K314100_OXT

Sequencing results for K314100_OXT

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