9. 100 mM NiSO 4 .
10. 50 mM ethylenediaminetetraacetic acid (EDTA).
11. 1 M guanidinium hydrochloride.
12. 20% ethanol.
13. Sodium dodecyl sulfate.
14. 30% acrylamide–bis solution, 29:1.
15. Ammonium persulfate (APS).
16. 10Â Tris–glycine–SDS (25 mM Tris, 192 mM glycine, 0.1%
SDS) at pH 8.3 for the resolving gel.
17. Tris–HCl (0.5 M, pH 6.8) for the stacking gel.
18. N,N,N,N-Tetramethylethylene-diamine (TEMED).
19. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 at pH of 7.4.
2.4 Assay Materials
1. Flat Bottom 96-well plates.
2. Synergy 4 Multi-Mode Microplate Reader (BioTek Instruments Inc.).
3. Concentrated sulfuric acid.
4. Autoclave bags.
5. Phenol.
3 Methods
3.1 Production
of Recombinant GFP
1. BL21(DE3) E. coli containing the GFP plasmid is inoculated
into 5 mL of LB media containing ampicillin and grown for
16–20 h at 37
C in a shaker set to 225 rpm.
2. One milliliter of the bacterial culture is then transferred into
1 L of LB media containing fresh ampicillin and shaken at
37
C until an OD 600nm of 0.4–0.5 is reached (approximately
3 h).
3. Protein expression is induced by supplementing the media with
100 μM IPTG. Afterward, the culture is incubated in a shaker
set to 225 rpm and 22
C for 20 h.
4. The cell culture is then harvested via centrifugation for 20 min
at 4000 rpm and 4
C (Sorvall Biofuge Primo R Centrifuge).
5. After removing the supernatant, the cell pellet is resuspended
in 20 mL of buffer A and placed on ice.
6. The cells are then lysed using sonication (Model CL-18, Fisher
Scientific) at an amplitude of 40–50. Cells are sonicated 10 s on
and 30 s off for 10 cycles.
480
Roozbeh Nayerhoda et al.
10. 50 mM ethylenediaminetetraacetic acid (EDTA).
11. 1 M guanidinium hydrochloride.
12. 20% ethanol.
13. Sodium dodecyl sulfate.
14. 30% acrylamide–bis solution, 29:1.
15. Ammonium persulfate (APS).
16. 10Â Tris–glycine–SDS (25 mM Tris, 192 mM glycine, 0.1%
SDS) at pH 8.3 for the resolving gel.
17. Tris–HCl (0.5 M, pH 6.8) for the stacking gel.
18. N,N,N,N-Tetramethylethylene-diamine (TEMED).
19. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 at pH of 7.4.
2.4 Assay Materials
1. Flat Bottom 96-well plates.
2. Synergy 4 Multi-Mode Microplate Reader (BioTek Instruments Inc.).
3. Concentrated sulfuric acid.
4. Autoclave bags.
5. Phenol.
3 Methods
3.1 Production
of Recombinant GFP
1. BL21(DE3) E. coli containing the GFP plasmid is inoculated
into 5 mL of LB media containing ampicillin and grown for
16–20 h at 37
C in a shaker set to 225 rpm.
2. One milliliter of the bacterial culture is then transferred into
1 L of LB media containing fresh ampicillin and shaken at
37
C until an OD 600nm of 0.4–0.5 is reached (approximately
3 h).
3. Protein expression is induced by supplementing the media with
100 μM IPTG. Afterward, the culture is incubated in a shaker
set to 225 rpm and 22
C for 20 h.
4. The cell culture is then harvested via centrifugation for 20 min
at 4000 rpm and 4
C (Sorvall Biofuge Primo R Centrifuge).
5. After removing the supernatant, the cell pellet is resuspended
in 20 mL of buffer A and placed on ice.
6. The cells are then lysed using sonication (Model CL-18, Fisher
Scientific) at an amplitude of 40–50. Cells are sonicated 10 s on
and 30 s off for 10 cycles.
480
Roozbeh Nayerhoda et al.