23. 1 M Tris–HCl, 1 L: Dissolve 121.1 g of Tris base in 800 mL of
H 2 O. Adjust the pH to 7.6 by adding concentrated HCl (~
60 mL). Adjust volume to 1 liter with ddH 2 O. Sterilize by
autoclaving (15 min).
24. 1 M MgCl 2 , 100 mL: Mix 20.33 g MgCl 2 with 70 mL of
ddH 2 O, add ddH 2 O until final volume is 100 mL. Sterilize
by autoclaving (15 min).
25. 5 M NaCl, 1 L: Mix 292.2 g of NaCl with 900 mL of ddH 2 O
by stirring, add ddH 2 O until final volume is 1 L. Sterilize by
autoclaving (15 min).
26. Buffer A. Modified Resuspension Buffer: 50 mM Tris–HCl
pH 7.6, 250 mM NaCl. Autoclave for long-term storage.
27. Buffer B. (For cell resuspension) made on day of use: To
100 mL of Buffer A add 120 mg of AEBSF, 50 μL of 1 M
DTT, 15 mg of lysozyme. Store on ice until used.
28. Buffer C. Resuspension Buffer with 5% glycerol (100 mL):
50 mM Tris–HCl pH 7.6, 300 mM NaCl, 5% glycerol.
29. Buffer D. ATP Wash Buffer (6 mL): 40 mM Tris–HCl pH 7.6,
200 mM NaCl, 5 mM MgCl 2 , 5% Glycerol. Add fresh immediately before use: 16.5 mg of ATP as a powder, 0.6 μL of 1 M
DTT, 30 μL of 10% DDM. Adjust to 6 mL with ddH 2 O. ATP
wash buffer includes ATP and Mg to remove any associated
chaperones.
30. Buffer E. Elution Buffer (10 mL): 25 mM Tris–HCl pH 7.6,
200 mM NaCl, 5% Glycerol.
Add fresh immediately before use: 1 μL of 1 M DTT, 50 μL
of 10% DDM, 8 mL of 0.25 M histidine pH 7.1. Adjust to
10 mL with ddH 2 O. 0.25 M L-Histidine (3.88 g/100 mL).
Make fresh; at room temperature will crystalize and at room
temperature will turn yellow. No need to pH.
31. Buffer F. Optional dialysis buffer (2 L, store and use at 4
C):
20 mM Tris–HCl pH 7.6, 20 mM NaCl, 5% glycerol. Add fresh
immediately before use: 0.4 g of DDM, 0.2 mL of 1 M DTT.
3 Methods
DAY 1
1. To 25 mL of 2Â YT medium, add 25 μL of ampicillin stock for
a final concentration of 50 μg/mL.
2. Inoculate with pMCSG9 Z -ISO E2 in C43 (DE3) cells
[5]. Grow overnight with shaking at 37
C.
DAY 2
56
Eleanore T. Wurtzel and Jesu ´ s Beltra ´ n
H 2 O. Adjust the pH to 7.6 by adding concentrated HCl (~
60 mL). Adjust volume to 1 liter with ddH 2 O. Sterilize by
autoclaving (15 min).
24. 1 M MgCl 2 , 100 mL: Mix 20.33 g MgCl 2 with 70 mL of
ddH 2 O, add ddH 2 O until final volume is 100 mL. Sterilize
by autoclaving (15 min).
25. 5 M NaCl, 1 L: Mix 292.2 g of NaCl with 900 mL of ddH 2 O
by stirring, add ddH 2 O until final volume is 1 L. Sterilize by
autoclaving (15 min).
26. Buffer A. Modified Resuspension Buffer: 50 mM Tris–HCl
pH 7.6, 250 mM NaCl. Autoclave for long-term storage.
27. Buffer B. (For cell resuspension) made on day of use: To
100 mL of Buffer A add 120 mg of AEBSF, 50 μL of 1 M
DTT, 15 mg of lysozyme. Store on ice until used.
28. Buffer C. Resuspension Buffer with 5% glycerol (100 mL):
50 mM Tris–HCl pH 7.6, 300 mM NaCl, 5% glycerol.
29. Buffer D. ATP Wash Buffer (6 mL): 40 mM Tris–HCl pH 7.6,
200 mM NaCl, 5 mM MgCl 2 , 5% Glycerol. Add fresh immediately before use: 16.5 mg of ATP as a powder, 0.6 μL of 1 M
DTT, 30 μL of 10% DDM. Adjust to 6 mL with ddH 2 O. ATP
wash buffer includes ATP and Mg to remove any associated
chaperones.
30. Buffer E. Elution Buffer (10 mL): 25 mM Tris–HCl pH 7.6,
200 mM NaCl, 5% Glycerol.
Add fresh immediately before use: 1 μL of 1 M DTT, 50 μL
of 10% DDM, 8 mL of 0.25 M histidine pH 7.1. Adjust to
10 mL with ddH 2 O. 0.25 M L-Histidine (3.88 g/100 mL).
Make fresh; at room temperature will crystalize and at room
temperature will turn yellow. No need to pH.
31. Buffer F. Optional dialysis buffer (2 L, store and use at 4
C):
20 mM Tris–HCl pH 7.6, 20 mM NaCl, 5% glycerol. Add fresh
immediately before use: 0.4 g of DDM, 0.2 mL of 1 M DTT.
3 Methods
DAY 1
1. To 25 mL of 2Â YT medium, add 25 μL of ampicillin stock for
a final concentration of 50 μg/mL.
2. Inoculate with pMCSG9 Z -ISO E2 in C43 (DE3) cells
[5]. Grow overnight with shaking at 37
C.
DAY 2
56
Eleanore T. Wurtzel and Jesu ´ s Beltra ´ n