91
1. Python 2.7.11 (www.python.org).
2. Bash 4.2.46 (www.gnu.org/software/bash/).
3. GROMACS 5.1.2 (www.gromacs.org) [6].
4. MARTINI 2.1 (http://md.chem.rug.nl/) [7].
5. PyMol 1.7.6.0 (https://sourceforge.net/projects/pymol).
6. GAWK 4.0.2 (www.gnu.org/software/gawk/).
7. DSSP 2.2.1 (http://swift.cmbi.ru.nl/gv/dssp/).
8. Grep 2.5.1 (https://www.gnu.org/software/grep/).
9. Curl 7.29.0 (https://curl.haxx.se/).
1. The gene of interest in an expression vector (see Subheading 3.3).
2. Primers containing the mutation of interest (see Subheading 3.3).
3. High-fidelity DNA polymerase kit.
4. Gibson assembly kit.
5. Thin-walled PCR tubes.
6. PCR thermocycler.
7. PCR purification kit.
8. Competent cells for cloning (Top10).
9. Competent cells for protein expression (BL21DE3).
10. Materials for colony PCR and analysis by gel electrophoresis:
(a) PCR master mix.
(b) T7 primers.
(c) 1% agarose gel made with SB buffer (46 g/L boric acid,
8 g/L sodium hydroxide), with a visualizing stain.
(d) DNA ladder mix.
(e) Agarose gel electrophoresis apparatus.
1. Buffer solution, Phosphate buffer (see Note 1): 0.05 M Sodium
phosphate, 0.2 M NaCl. Adjust the pH to what is appropriate
for both the protein of interest and the chemistry that is needed
to label the protein with the synthetic dye (using either hydrochloric acid or sodium hydroxide).
2. TCEP stock solution: dissolve 0.1437 g of Tris(2-carboxyethyl)
phosphine hydrochloride (TCEP-HCl) in 1 mL of buffer
solution (501 mM stock solution) (see Note 2).
3. Dye stock solution: Add a suitable solvent to the synthetic dye
to achieve a stock solution with a final concentration of
10 mM. In the case of the Alexa Fluor 532 C5 Maleimide,
1 mg was dissolved in 123 μL of phosphate buffer (10 mM
stock solution) (see Note 3).
2.1 Software
2.2 Cloning
and Protein
Purification
Components
2.3 Dye Labeling
Components
Synthetic-Dye Fluorescent Protein FRET Sensors
1. Python 2.7.11 (www.python.org).
2. Bash 4.2.46 (www.gnu.org/software/bash/).
3. GROMACS 5.1.2 (www.gromacs.org) [6].
4. MARTINI 2.1 (http://md.chem.rug.nl/) [7].
5. PyMol 1.7.6.0 (https://sourceforge.net/projects/pymol).
6. GAWK 4.0.2 (www.gnu.org/software/gawk/).
7. DSSP 2.2.1 (http://swift.cmbi.ru.nl/gv/dssp/).
8. Grep 2.5.1 (https://www.gnu.org/software/grep/).
9. Curl 7.29.0 (https://curl.haxx.se/).
1. The gene of interest in an expression vector (see Subheading 3.3).
2. Primers containing the mutation of interest (see Subheading 3.3).
3. High-fidelity DNA polymerase kit.
4. Gibson assembly kit.
5. Thin-walled PCR tubes.
6. PCR thermocycler.
7. PCR purification kit.
8. Competent cells for cloning (Top10).
9. Competent cells for protein expression (BL21DE3).
10. Materials for colony PCR and analysis by gel electrophoresis:
(a) PCR master mix.
(b) T7 primers.
(c) 1% agarose gel made with SB buffer (46 g/L boric acid,
8 g/L sodium hydroxide), with a visualizing stain.
(d) DNA ladder mix.
(e) Agarose gel electrophoresis apparatus.
1. Buffer solution, Phosphate buffer (see Note 1): 0.05 M Sodium
phosphate, 0.2 M NaCl. Adjust the pH to what is appropriate
for both the protein of interest and the chemistry that is needed
to label the protein with the synthetic dye (using either hydrochloric acid or sodium hydroxide).
2. TCEP stock solution: dissolve 0.1437 g of Tris(2-carboxyethyl)
phosphine hydrochloride (TCEP-HCl) in 1 mL of buffer
solution (501 mM stock solution) (see Note 2).
3. Dye stock solution: Add a suitable solvent to the synthetic dye
to achieve a stock solution with a final concentration of
10 mM. In the case of the Alexa Fluor 532 C5 Maleimide,
1 mg was dissolved in 123 μL of phosphate buffer (10 mM
stock solution) (see Note 3).
2.1 Software
2.2 Cloning
and Protein
Purification
Components
2.3 Dye Labeling
Components
Synthetic-Dye Fluorescent Protein FRET Sensors