Biomod/2013/StJohns/results: Difference between revisions
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We have | =Summary= | ||
*We have synthesized versions of the claw with (‘sticky’) and without (‘blunt’) single-stranded binding elements. | |||
*We have synthesized versions of the claw with and without fluorescent tags for FRET analysis. | |||
*We have visualized the above versions of the claw on AFM to show that they take the predicted shape. | |||
*We have shown that the above versions of the claw form tight bands on a gel, indicating a single primary product of the anneal. | |||
*We have demonstrated a binding interaction between the functionalized claw and functionalized capsid as well as a lack of interaction between the nonfunctionalized claw and capsid. | |||
*We have not been able to differentiate bound and unbound complexes via DLS. | |||
*We have generated and isolated FAB fragments for use as binding elements in future claw designs. | |||
*We have demonstrated the potential for selecting claws based on their avidity using a chromatography augmented with photocleavable elements. | |||
Below are AFM images of the 100nm armed claws. | |||
<html><center><table><tbody align="center"><tr><td><img src="http://openwetware.org/images/thumb/6/66/Lukemanlab-Bluntclaw_afm.png/200px-Lukemanlab-Bluntclaw_afm.png"></td><td><img src="http://openwetware.org/images/thumb/9/9f/Lukemanlab-Stickyclaw_afm.png/200px-Lukemanlab-Stickyclaw_afm.png"></td></tr><tr><td>"Blunt" claw</td><td>"Sticky" claw</td></tr></tbody></table></center></html> | <html><center><table><tbody align="center"><tr><td><img src="http://openwetware.org/images/thumb/6/66/Lukemanlab-Bluntclaw_afm.png/200px-Lukemanlab-Bluntclaw_afm.png"></td><td><img src="http://openwetware.org/images/thumb/9/9f/Lukemanlab-Stickyclaw_afm.png/200px-Lukemanlab-Stickyclaw_afm.png"></td></tr><tr><td>"Blunt" claw</td><td>"Sticky" claw</td></tr></tbody></table></center></html> | ||
Revision as of 11:56, 24 October 2013
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Summary
- We have synthesized versions of the claw with (‘sticky’) and without (‘blunt’) single-stranded binding elements.
- We have synthesized versions of the claw with and without fluorescent tags for FRET analysis.
- We have visualized the above versions of the claw on AFM to show that they take the predicted shape.
- We have shown that the above versions of the claw form tight bands on a gel, indicating a single primary product of the anneal.
- We have demonstrated a binding interaction between the functionalized claw and functionalized capsid as well as a lack of interaction between the nonfunctionalized claw and capsid.
- We have not been able to differentiate bound and unbound complexes via DLS.
- We have generated and isolated FAB fragments for use as binding elements in future claw designs.
- We have demonstrated the potential for selecting claws based on their avidity using a chromatography augmented with photocleavable elements.
Below are AFM images of the 100nm armed claws.
<html><center><table><tbody align="center"><tr><td><img src="http://openwetware.org/images/thumb/6/66/Lukemanlab-Bluntclaw_afm.png/200px-Lukemanlab-Bluntclaw_afm.png"></td><td><img src="http://openwetware.org/images/thumb/9/9f/Lukemanlab-Stickyclaw_afm.png/200px-Lukemanlab-Stickyclaw_afm.png"></td></tr><tr><td>"Blunt" claw</td><td>"Sticky" claw</td></tr></tbody></table></center></html>
Additionally, we have used gel electrophoresis to characterize the binding of Sticky/Blunt DO to Sticky/WT capsids.
We use FRET as a molecular ruler to measure the distance between the claw and capsid during binding.
The following gel images demonstrate FRET fluoresnce.
<html> <center> <table> <tbody align="center"> <tr> <td> <img src="http://openwetware.org/images/thumb/d/d5/1a_ch1_FRET.tif/345px-1a_ch1_FRET.tif.png"> </td> <td> <img src="http://openwetware.org/images/thumb/6/69/1a_ch2_DONOR.tif/345px-1a_ch2_DONOR.tif.png"> </td> <td> <img src="http://openwetware.org/images/thumb/c/ce/1a_ch3_ACCEPTOR.tif/345px-1a_ch3_ACCEPTOR.tif.png"> </td> </tr> <tr> <td>FRET</td><td>DONOR</td><td>ACCEPTOR</td> </tr> </tbody> </table> </center> </html>
To determine FRET, FRET intensity - DONOR intensity - ACCEPTOR intensity
DA(i,j) = DA(i,j) - beta_leak * DD(i,j) - beta_dir * AA (i,j)