110
7. Ni
2+
-NTA agarose slurry.
8. Propylene column.
9. Ni
2+
-NTA lysis buffer: 50 mM KH 2 PO 4 , pH 8, 150 mM NaCl,
5 mM imidazole.
10. Phenylmethylsulfonyl fluoride (PMSF): 100 mM in ethanol.
11. Lysozyme: 10 mg/mL in water.
12. Ni
2+
-NTA wash buffer: 50 mM KH 2 PO 4 , pH 7.5, 150 mM
NaCl, 10 mM imidazole.
13. Ni
2+
-NTA elution buffer: 50 mM KH 2 PO 4 , pH 7.5, 150 mM
NaCl, 500 mM imidazole.
14. Bradford reagent.
15. Streptavidin-resin in propylene column, such as the StrepTactin column.
16. Streptavidin wash buffer: 100 mM Tris–HCl, pH 8, 150 mM
NaCl, 1 mM EDTA.
17. Streptavidin elution buffer 100 mM Tris–HCl, pH 8, 150 mM
NaCl, 1 mM EDTA, 2.5 mM desthiobiotin.
18. Centrifugal filter device with appropriate molecular weight
cutoff.
19. Storage buffer: 50 mM HEPES, 50 mM NaCl, pH 7.2.
1. Labeling buffer: 50 mM HEPES, 50 mM NaCl, pH 7.2,
1 mg/mL bovine serum albumin (BSA).
2. Fluorescein or tetramethylrhodamine (TMR) labeled ligand
stock solution in DMSO, for characterizing the binding
strength by means of fluorescence polarization.
3. BG-fluorophore-ligand conjugate stock solution in DMSO, for
labeling SNAP-tag based sensors with an intramolecular tether.
4. BC-fluorophore-ligand conjugate stock solution in DMSO, for
labeling CLIP-tag-based sensors with an intramolecular tether.
5. Nonbinding multiwell plates (black or white).
6. Plate reader with filters for fluorescent polarization and fluorescent measurements.
7. Nano-glo
®
substrate from Promega.
1. Glass coverslips (Ø 15 mm).
2. Ethanol.
3. Poly-L-lysine solution: 0.7 mg/mL in sterile water.
4. HEK293 cells.
5. Lipofectamine 2000.
6. Opti-MEM
®
I reduced serum medium.
2.2 In Vitro Labeling
and Characterization
2.3 Cell-Surface
Expression
Helen Farrants et al.