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Team:TU Eindhoven/Characterization/UVvis
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<h2>Characterization of pAzF</h2> | <h2>Characterization of pAzF</h2> | ||
| + | <figure style="float:right;margin-right:0;"> | ||
| + | <img id='Fig3' src="https://static.igem.org/mediawiki/2014/5/5b/TU_Eindhoven_Non-exposed_pAzF_%2B_DBCO.jpg" width="500" style="display: inline-block; border: 4px solid #00BAC6; padding: 4px; background: #222; margin-bottom: 10px;"> | ||
| + | <figcaption style="font-size:18px;color:#CCCCCC;">Figure 1. Graph showing absorption at 308.5 nm over time with non-<br />exposed pAzF</figcaption> | ||
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<p>p-Azdio-L-phenylalanine is a photocrosslinking agent. Upon irradiation with UV light at wavelengths below 310 nm, simple aryl-azides extrude molecular nitrogen to form short-lived (~ 1 ns) singlet nitrenes which rearrange to form dehydroazepines, these react rapidly with amines to form robust adducts. [1] | <p>p-Azdio-L-phenylalanine is a photocrosslinking agent. Upon irradiation with UV light at wavelengths below 310 nm, simple aryl-azides extrude molecular nitrogen to form short-lived (~ 1 ns) singlet nitrenes which rearrange to form dehydroazepines, these react rapidly with amines to form robust adducts. [1] | ||
| - | To characterize the sensitivity of pAzF to light, the SPAAC reaction of pAzF and DBCO- | + | To characterize the sensitivity of pAzF to light, the SPAAC reaction of pAzF and DBCO-PEG<sub>4</sub>-NHS ester was monitored twice using UV-vis. One reaction was performed using a pAzF solution that was not exposed to light. The other reaction was performed with a pAzF solution that had been irradiated by a light source (200 – 400 nm) for sixteen hours. Check our protocol <a href="https://static.igem.org/mediawiki/2014/1/1d/TU_Eindhoven_Protocol_Monitoring_SPAAC_with_UVVIS.pdf">here</a>. |
| - | The absorption spectrum of DBCO- | + | The absorption spectrum of DBCO-PEG<sub>4</sub>-NHS ester has a peak at 308.5 nm. When the DBCO reacts with pAzF, this absorption will decrease.</p> |
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| + | <figure style="float:left;margin-left:0;"> | ||
| + | <img id='Fig3' src="https://static.igem.org/mediawiki/2014/b/b1/TU_Eindhoven_Exposed_pAzF_%2B_DBCO.jpg" width="500" style="display: inline-block; border: 4px solid #00BAC6; padding: 4px; background: #222; margin-bottom: 10px;"> | ||
| + | <figcaption style="font-size:18px;color:#CCCCCC;">Figure 2. Graph showing absorption at 308.5 nm over time with pre-<br />exposed pAzF</figcaption> | ||
| + | </figure> | ||
<p>Both graphs show a decrease in absorbance at the 308.5 nm wavelength, this means that triple bond in DBCO has reacted. Since this is only possible with the azide group this must mean that the pAzF is still reactive enough even after prolonged exposure to short wavelength light (200 – 400 nm).</p> | <p>Both graphs show a decrease in absorbance at the 308.5 nm wavelength, this means that triple bond in DBCO has reacted. Since this is only possible with the azide group this must mean that the pAzF is still reactive enough even after prolonged exposure to short wavelength light (200 – 400 nm).</p> | ||
| - | <h4>Bibliography</h4> | + | <h4>Bibliography</h4><p> |
[1] Chin J.W., Santoro S.W., Martin A.B., King D.S., Wang L. and Schultz P.G., (2002), Addition of p-Azido-L-phenylalanine to the Genetic Code of Escherichia coli, <i>JACS</i>, 124, 9026-9027 | [1] Chin J.W., Santoro S.W., Martin A.B., King D.S., Wang L. and Schultz P.G., (2002), Addition of p-Azido-L-phenylalanine to the Genetic Code of Escherichia coli, <i>JACS</i>, 124, 9026-9027 | ||
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Latest revision as of 23:19, 17 October 2014
Characterization of pAzF
exposed pAzF
p-Azdio-L-phenylalanine is a photocrosslinking agent. Upon irradiation with UV light at wavelengths below 310 nm, simple aryl-azides extrude molecular nitrogen to form short-lived (~ 1 ns) singlet nitrenes which rearrange to form dehydroazepines, these react rapidly with amines to form robust adducts. [1] To characterize the sensitivity of pAzF to light, the SPAAC reaction of pAzF and DBCO-PEG4-NHS ester was monitored twice using UV-vis. One reaction was performed using a pAzF solution that was not exposed to light. The other reaction was performed with a pAzF solution that had been irradiated by a light source (200 – 400 nm) for sixteen hours. Check our protocol here. The absorption spectrum of DBCO-PEG4-NHS ester has a peak at 308.5 nm. When the DBCO reacts with pAzF, this absorption will decrease.
exposed pAzF
Both graphs show a decrease in absorbance at the 308.5 nm wavelength, this means that triple bond in DBCO has reacted. Since this is only possible with the azide group this must mean that the pAzF is still reactive enough even after prolonged exposure to short wavelength light (200 – 400 nm).
Bibliography
[1] Chin J.W., Santoro S.W., Martin A.B., King D.S., Wang L. and Schultz P.G., (2002), Addition of p-Azido-L-phenylalanine to the Genetic Code of Escherichia coli, JACS, 124, 9026-9027
