7. Pierce NeutrAvidin-coated, high binding capacity, eight-well
strips (ThermoFisher Scientific, catalog no. 15508).
8. Dynabeads MyOne Streptavidin T1 (ThermoFisher Scientific,
catalog no. 65601 or 65602).
9. Eppendorf LoBind microcentrifuge tubes (Eppendorf, catalog
no. 0030 108.116).
10. Magnetic separation rack for 1.5-ml microcentrifuge tubes—
for example, DynaMag™-2 Magnet (ThermoFisher Scientific,
catalog no. 12321D).
11. PBS: Prepare a 10Â stock by dissolving 80.0 g of NaCl
(MW ¼ 58.44), 2.0 g of KCl (MW ¼ 74.55), 14.4 g of
Na 2 HPO 4 (MW ¼ 141.96), and 2.4 g of KH 2 PO 4
(MW ¼ 136.09) in 800 ml of dH 2 O. Adjust the pH to 7.4
with HCl and add dH 2 O until a total volume of 1 l. Autoclave
to sterilize and store at room temperature. For a 1Â stock,
dilute the 10Â stock with dH 2 O and adjust the pH to 7.4
with HCl.
12. PBST: Dilute the 10Â PBS to 1Â with dH 2 O and 0.1% (v/v)
Tween 20, and adjust to pH 7.4 with HCl.
13. 10Â Casein Blocking Buffer (Sigma-Aldrich, catalog
no. B6429).
14. 2Â Blocking Buffer: Dilute 10Â Casein Blocking Buffer fivefold in PBS.
15. 2YT medium: Dissolve 16 g of Bacto tryptone, 10 g of Bacto
yeast extract, and 5 g of NaCl (MW ¼ 58.44) in 900 ml of
dH 2 O. Adjust the pH to 7.0 with NaOH and add dH 2 O until a
total volume of 1 l. Autoclave to sterilize and store at room
temperature.
16. Tetracycline hydrochloride (1000Â stock): Dissolve tetracycline hydrochloride (12 mg/ml) in 70% ethanol. Store at
À20
C, protected from light. Working concentration is
12 μg/ml.
17. 0.2 M glycine, pH 2.2: Dissolve 1.5 g of glycine
(MW ¼ 75.07) in 80 ml of dH 2 O. Adjust the pH to 2.2 with
HCl and add dH 2 O until a total volume of 100 ml. Autoclave
to sterilize and store at room temperature.
18. 1 M Tris-HCl, pH 9.1: Dissolve 121.14 g of Tris base
(MW ¼ 121.14) in 800 ml of dH 2 O. Adjust the pH to 9.1
with HCl and add dH 2 O until a total volume of 1 l. The pH of
Tris buffers varies with temperature and concentration; therefore, adjust the pH at room temperature. Autoclave to sterilize
and store at room temperature.
19. Triethylamine: Dilute 14 μl of triethylamine (Sigma-Aldrich,
catalog no. T0886) in 986 μl of PBS, immediately before use.
108
Anna A. S. Tang et al.
strips (ThermoFisher Scientific, catalog no. 15508).
8. Dynabeads MyOne Streptavidin T1 (ThermoFisher Scientific,
catalog no. 65601 or 65602).
9. Eppendorf LoBind microcentrifuge tubes (Eppendorf, catalog
no. 0030 108.116).
10. Magnetic separation rack for 1.5-ml microcentrifuge tubes—
for example, DynaMag™-2 Magnet (ThermoFisher Scientific,
catalog no. 12321D).
11. PBS: Prepare a 10Â stock by dissolving 80.0 g of NaCl
(MW ¼ 58.44), 2.0 g of KCl (MW ¼ 74.55), 14.4 g of
Na 2 HPO 4 (MW ¼ 141.96), and 2.4 g of KH 2 PO 4
(MW ¼ 136.09) in 800 ml of dH 2 O. Adjust the pH to 7.4
with HCl and add dH 2 O until a total volume of 1 l. Autoclave
to sterilize and store at room temperature. For a 1Â stock,
dilute the 10Â stock with dH 2 O and adjust the pH to 7.4
with HCl.
12. PBST: Dilute the 10Â PBS to 1Â with dH 2 O and 0.1% (v/v)
Tween 20, and adjust to pH 7.4 with HCl.
13. 10Â Casein Blocking Buffer (Sigma-Aldrich, catalog
no. B6429).
14. 2Â Blocking Buffer: Dilute 10Â Casein Blocking Buffer fivefold in PBS.
15. 2YT medium: Dissolve 16 g of Bacto tryptone, 10 g of Bacto
yeast extract, and 5 g of NaCl (MW ¼ 58.44) in 900 ml of
dH 2 O. Adjust the pH to 7.0 with NaOH and add dH 2 O until a
total volume of 1 l. Autoclave to sterilize and store at room
temperature.
16. Tetracycline hydrochloride (1000Â stock): Dissolve tetracycline hydrochloride (12 mg/ml) in 70% ethanol. Store at
À20
C, protected from light. Working concentration is
12 μg/ml.
17. 0.2 M glycine, pH 2.2: Dissolve 1.5 g of glycine
(MW ¼ 75.07) in 80 ml of dH 2 O. Adjust the pH to 2.2 with
HCl and add dH 2 O until a total volume of 100 ml. Autoclave
to sterilize and store at room temperature.
18. 1 M Tris-HCl, pH 9.1: Dissolve 121.14 g of Tris base
(MW ¼ 121.14) in 800 ml of dH 2 O. Adjust the pH to 9.1
with HCl and add dH 2 O until a total volume of 1 l. The pH of
Tris buffers varies with temperature and concentration; therefore, adjust the pH at room temperature. Autoclave to sterilize
and store at room temperature.
19. Triethylamine: Dilute 14 μl of triethylamine (Sigma-Aldrich,
catalog no. T0886) in 986 μl of PBS, immediately before use.
108
Anna A. S. Tang et al.