99
13. There are two conserved cysteine residues in the GFP family;
we have had success with the mutations C49S and C71V with
minimal fluorescence loss, as described by Suzuki et al. [8].
14. The reaction volume and reagents should be scaled appropriately,
relative to the concentration of the protein stock solution.
15. When agitating the solution, care should be made that the
solution does not begin to form froth or foam as this can lead
to precipitation of protein.
16. Filtration can be used instead of centrifugation; however, there is
typically some volume/yield loss when filtering small volumes.
17. PD-10 columns (GE healthcare) are usually sufficient to separate free dye from the protein. If the purity of the protein is a
concern, the protein should be first buffer exchanged to
remove excess TCEP and then purified with SEC (GE healthcare, Hiload 26/600 superdex 200pg, adequate for most proteins). This should separate labeled protein from free dye and
any contaminant proteins.
References
1. Scanziani M, Hausser M (2009) Electrophysiology
in the age of light. Nature 461(7266):930–939
2. Hou B-H, Takanaga H, Grossmann G, Chen
L-Q, Qu X-Q, Jones AM, Lalonde S, Schweissgut
O, Wiechert W, Frommer WB (2011) Optical sensors for monitoring dynamic changes of intracellular metabolite levels in mammalian cells. Nat
Protoc 6(11):1818–1833. http://www.nature.
c o m / n p r o t / j o u r n a l / v 6 / n 1 1 / a b s /
n p r o t . 2 0 1 1 . 3 9 2 . h t m l # s u p p l e m e n t a r y -
information
3. Masharina A, Reymond L, Maurel D, Umezawa
K, Johnsson K (2012) A Fluorescent sensor for
GABA and synthetic GABAB receptor ligands.
J Am Chem Soc 134(46):19026–19034.
doi:10.1021/ja306320s
4. Namiki S, Sakamoto H, Iinuma S, Iino M,
Hirose K (2007) Optical glutamate sensor for
spatiotemporal analysis of synaptic transmission. Eur J Neurosci 25(8):2249–2259.
doi:10.1111/j.1460-9568.2007.05511.x
5. Suzuki M, Tanaka S, Ito Y, Inoue M, Sakai T,
Nishigaki K (2012) Simple and tunable Förster
resonance energy transfer-based bioprobes for
high-throughput monitoring of caspase-3 activation in living cells by using flow cytometry.
Biochim Biophys Acta 1823(2):215–226.
doi:10.1016/j.bbamcr.2011.07.006
6. Hess B, Kutzner C, Van Der Spoel D, Lindahl E
(2008) GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput 4(3):435–447
7. Monticelli L, Kandasamy SK, Periole X, Larson
RG, Tieleman DP, Marrink S-J (2008) The
MARTINI coarse-grained force field: extension to
proteins. J Chem Theory Comput 4(5):819–834
8. Suzuki T, Arai S, Takeuchi M, Sakurai C, Ebana
H, Higashi T, Hashimoto H, Hatsuzawa K,
Wada I (2012) Development of cysteine-free
fluorescent proteins for the oxidative environment. PLoS One 7(5):e37551. doi:10.1371/
journal.pone.0037551
Synthetic-Dye Fluorescent Protein FRET Sensors
13. There are two conserved cysteine residues in the GFP family;
we have had success with the mutations C49S and C71V with
minimal fluorescence loss, as described by Suzuki et al. [8].
14. The reaction volume and reagents should be scaled appropriately,
relative to the concentration of the protein stock solution.
15. When agitating the solution, care should be made that the
solution does not begin to form froth or foam as this can lead
to precipitation of protein.
16. Filtration can be used instead of centrifugation; however, there is
typically some volume/yield loss when filtering small volumes.
17. PD-10 columns (GE healthcare) are usually sufficient to separate free dye from the protein. If the purity of the protein is a
concern, the protein should be first buffer exchanged to
remove excess TCEP and then purified with SEC (GE healthcare, Hiload 26/600 superdex 200pg, adequate for most proteins). This should separate labeled protein from free dye and
any contaminant proteins.
References
1. Scanziani M, Hausser M (2009) Electrophysiology
in the age of light. Nature 461(7266):930–939
2. Hou B-H, Takanaga H, Grossmann G, Chen
L-Q, Qu X-Q, Jones AM, Lalonde S, Schweissgut
O, Wiechert W, Frommer WB (2011) Optical sensors for monitoring dynamic changes of intracellular metabolite levels in mammalian cells. Nat
Protoc 6(11):1818–1833. http://www.nature.
c o m / n p r o t / j o u r n a l / v 6 / n 1 1 / a b s /
n p r o t . 2 0 1 1 . 3 9 2 . h t m l # s u p p l e m e n t a r y -
information
3. Masharina A, Reymond L, Maurel D, Umezawa
K, Johnsson K (2012) A Fluorescent sensor for
GABA and synthetic GABAB receptor ligands.
J Am Chem Soc 134(46):19026–19034.
doi:10.1021/ja306320s
4. Namiki S, Sakamoto H, Iinuma S, Iino M,
Hirose K (2007) Optical glutamate sensor for
spatiotemporal analysis of synaptic transmission. Eur J Neurosci 25(8):2249–2259.
doi:10.1111/j.1460-9568.2007.05511.x
5. Suzuki M, Tanaka S, Ito Y, Inoue M, Sakai T,
Nishigaki K (2012) Simple and tunable Förster
resonance energy transfer-based bioprobes for
high-throughput monitoring of caspase-3 activation in living cells by using flow cytometry.
Biochim Biophys Acta 1823(2):215–226.
doi:10.1016/j.bbamcr.2011.07.006
6. Hess B, Kutzner C, Van Der Spoel D, Lindahl E
(2008) GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput 4(3):435–447
7. Monticelli L, Kandasamy SK, Periole X, Larson
RG, Tieleman DP, Marrink S-J (2008) The
MARTINI coarse-grained force field: extension to
proteins. J Chem Theory Comput 4(5):819–834
8. Suzuki T, Arai S, Takeuchi M, Sakurai C, Ebana
H, Higashi T, Hashimoto H, Hatsuzawa K,
Wada I (2012) Development of cysteine-free
fluorescent proteins for the oxidative environment. PLoS One 7(5):e37551. doi:10.1371/
journal.pone.0037551
Synthetic-Dye Fluorescent Protein FRET Sensors