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I'd like to make a FRET sensor to detect changes in conformation of an intracellular protein. I'd like to use fluorescent proteins on the flanks, but I am not sure about the distance between the fluorescent proteins and my protein.
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Vibor Laketa
Head of Microscopy Unit | Infectious Diseases Imaging Platform (IDIP), University Hospital Heidelberg
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- Imaging Platform Coordinator (Deutsches Zentrum fur Infektionsforschung, DZIF) | Infectious Diseases University Hospital Heidelberg
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Alen Piljić
Managing director | Life Science Network gGmbH
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Alen Piljić Thursday, 04 July 2013 - 00:52 UTC
In principle you should try several linkers and also different circular permutations of the fluorescent proteins.
You can check out this publication:
http://www.lifescience.net/publications/3/rapid-development-of-genetically-encoded-fret-repo/Detailed cloning procedure is described here:
http://www.lifescience.net/protocols/42/cloning-of-ratiometric-fret-sensors-using-f-series/The plasmid library to start with can be obtained from Carsten Schultz, EMBL Heidelberg.