Team:Linkoping Sweden/Results

From 2014.igem.org

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<p>Our project idea is also based on the labeling of our antibodies with a green fluorescent probe called FITC. The labeling was performed by following the labeling protocol from Thermo Scientific and the result was analyzed by measuring the absorbance. In (Fig.7) one can see the result from the FITC labeled polyclonal antibodies in which the concentration of FITC at Amax (494 nm) was calculated to be 11.79 microM as well as the calculated protein concentration at A280 (277.5) turned out to be 5.6 microM, indicating a 2:1 ratio of labeling. In (Fig.8) one can see the result from the FITC labeled monoclonal antibodies in which the concentration of FITC at Amax (493 nm) was calculated to be 5.0 microM as well as the calculated protein concentration at A280 (278.5) turned out to be 4.7 microM, indicating a 1:1 ratio of labeling.  
<p>Our project idea is also based on the labeling of our antibodies with a green fluorescent probe called FITC. The labeling was performed by following the labeling protocol from Thermo Scientific and the result was analyzed by measuring the absorbance. In (Fig.7) one can see the result from the FITC labeled polyclonal antibodies in which the concentration of FITC at Amax (494 nm) was calculated to be 11.79 microM as well as the calculated protein concentration at A280 (277.5) turned out to be 5.6 microM, indicating a 2:1 ratio of labeling. In (Fig.8) one can see the result from the FITC labeled monoclonal antibodies in which the concentration of FITC at Amax (493 nm) was calculated to be 5.0 microM as well as the calculated protein concentration at A280 (278.5) turned out to be 4.7 microM, indicating a 1:1 ratio of labeling.  
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Our goal is to test our FRET effect by expressing our RFP/MCherry linked epitope protein before entering the competition in Boston. Hopefully we will be able to perform this before the deadline, otherwise we will continue with our project afterwards.
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Our goal is to test our FRET effect by expressing our RFP/MCherry linked epitope protein before entering the competition in Boston. Hopefully we will be able to perform this before the deadline, otherwise we will continue with our project afterwards.</p>
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(Bild på rör med inmärkta antikroppar samt en schematisk bild på en antikropp med inmärkt FITC).</p>
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<img src="https://static.igem.org/mediawiki/2014/thumb/1/18/Linkoping_sweden_FITC.jpg/449px-Linkoping_sweden_FITC.jpg" style="max-width:200px;max-height:240px;" title="Labeling of monoclonal and polyclonal antibodies with a green fluorescent probe (FITC)."></a>
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        <img src="https://static.igem.org/mediawiki/2014/1/18/Linkoping_sweden_FITC.jpg" title="Labeling of monoclonal and polyclonal antibodies with a green fluorescent probe (FITC)." style="background:#FFFFFF;max-width:100%;max-height:100%;">
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        <p>Fig 7. Labeling of monoclonal and polyclonal antibodies with a green fluorescent probe (FITC).</p>
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<p>Fig 7. Labeling of monoclonal and polyclonal antibodies with a green fluorescent probe (FITC).</p>
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<img src="https://static.igem.org/mediawiki/2014/thumb/5/5f/Linkoping_sweden_Antikropp-01-01.png/731px-Linkoping_sweden_Antikropp-01-01.png" style="max-width:370px;max-height:240px;" title="Schematic illustration of an antibody labeled with FITC."></a>
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        <img src="https://static.igem.org/mediawiki/2014/5/5f/Linkoping_sweden_Antikropp-01-01.png" title="Schematic illustration of an antibody labeled with FITC." style="background:#FFFFFF;max-width:100%;max-height:100%;">
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        <p>Fig 8. Schematic illustration of an antibody labeled with FITC.</p>
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<p>Fig 8. Schematic illustration of an antibody labeled with FITC.</p>
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Revision as of 10:39, 16 October 2014

Our vision is to create a biobrick including the sequence of the Ara h1 protein linked to a red fluorescent protein so that we in turn can express the protein and thus provide an interaction of this protein complex with the antibodies. To ensure ourselves that this epitope-red fluorescent protein complex will bind to the Ara h1 specific IgG antibodies several ideas of biobrick-setup was brought to mind. Since we use both monoclonal antibodies specific for epitope 2 of Ara h1 and polyclonal antibodies specific for several epitopes of Ara h1 we decided to create a biobrick consisting of epitope 2 of Ara h1 linked to a red fluorescent protein as well as a biobrick consisting of five wisely chosen epitopes (epitope 22, epitope 1, epitope 3, epitope 4, and epitope 17) of Ara h1 linked to a red fluorescent protein. However, since there are two different mutants of the red fluorescent protein (called RFP and MCherry) we decided to create two setups of every biobrick combination to ensure that the best possible detection by FRET is used since RFP and MCherry slightly differs in their wavelength areas. The main idea is to practically test this FRET effect in both epitope-RFP and epitope-MCherry combinations by fluorescence which hopefully will prove to us which mutant is best suited to use and thus prove that our theory actually works.

Linköping University
581 83 Linköping, Sweden
liuigemgroup@gmail.com
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