Difference between revisions of "20.109:Module 2"
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==Module 2== | ==Module 2== | ||
| − | '''Instructors:''' Alan Jasanoff and [ | + | '''Instructors:''' Alan Jasanoff and [http://openwetware.org/wiki/Natalie_Kuldell ] |
| − | '''TA:''' [ | + | '''TA:''' [http://openwetware.org/wiki/Yoon_Sung_Nam ] |
| − | Once proteins are synthesized, their localization and conformation are critical to their function, but without | + | Once proteins are synthesized, their localization and conformation are critical to their function, but without X-ray vision to peer into a cell, these can be hard to detect. Fluorescence is one of the best tools in our toolkit for measuring proteins in living cells, allowing cellular functions to be probed unobtrusively. In this experimental module we will measure calcium fluctuations in living cells by tagging a calcium sensing protein with a fluorescent one. In the presence of calcium, the fluorescent protein will fold and the cells should appear green. We can transfect the genetically encoded calcium sensor into cells of interest, in this case mouse embryonic stem cells. Changes in fluorescence will allow us to quantitatively measure how chemical and physical perturbations affect calcium signaling in these cells. |
| − | [[Image:Macintosh HD-Users-nkuldell-Desktop- | + | [[Image:Macintosh HD-Users-nkuldell-Desktop-109S07 mod3 coverart.png|thumb|500px|center|Calmodulin structure visualized with Cn3D for PDB file 1DMO<br>Mouse embryonic stem cells expressing genetically-encoded Ca2+ sensor<br> images from N. Kuldell]] |
[[20.109(S07): Start-up signal measurement| Module 2 Day 1: Start-up signal measurement]]<br> | [[20.109(S07): Start-up signal measurement| Module 2 Day 1: Start-up signal measurement]]<br> | ||
| Line 17: | Line 17: | ||
[[20.109(S07): Lipofection| Module 2 Day 3: Lipofection]]<br> | [[20.109(S07): Lipofection| Module 2 Day 3: Lipofection]]<br> | ||
[[20.109(S07): Measuring calcium in vivo| Module 2 Day 4: Measuring calcium in vivo]]<br> | [[20.109(S07): Measuring calcium in vivo| Module 2 Day 4: Measuring calcium in vivo]]<br> | ||
| − | [[20.109(S07): Calcium signaling in vivo| Module 2 Day 5:Calcium signaling in vivo]]<br> | + | [[20.109(S07): Calcium signaling in vivo| Module 2 Day 5: Calcium signaling in vivo]]<br> |
[[20.109(S07): Student presentations, module 2 | Module 2 Day 6: Student presentations]]<br> | [[20.109(S07): Student presentations, module 2 | Module 2 Day 6: Student presentations]]<br> | ||
| − | [ | + | [http://openwetware.org/wiki/20.109(S07):_TA's_notes_for_module_2 | TA notes, mod 2] |
Latest revision as of 15:31, 15 June 2015
Module 2
Instructors: Alan Jasanoff and [1]
TA: [2]
Once proteins are synthesized, their localization and conformation are critical to their function, but without ���X-ray vision��� to peer into a cell, these can be hard to detect. Fluorescence is one of the best tools in our toolkit for measuring proteins in living cells, allowing cellular functions to be probed unobtrusively. In this experimental module we will measure calcium fluctuations in living cells by tagging a calcium sensing protein with a fluorescent one. In the presence of calcium, the fluorescent protein will fold and the cells should appear green. We can transfect the genetically encoded calcium sensor into cells of interest, in this case mouse embryonic stem cells. Changes in fluorescence will allow us to quantitatively measure how chemical and physical perturbations affect calcium signaling in these cells.
Mouse embryonic stem cells expressing genetically-encoded Ca2+ sensor
images from N. Kuldell
Module 2 Day 1: Start-up signal measurement
Module 2 Day 2: Measuring calcium in vitro
Module 2 Day 3: Lipofection
Module 2 Day 4: Measuring calcium in vivo
Module 2 Day 5: Calcium signaling in vivo
Module 2 Day 6: Student presentations
