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Comparative Physiology @ Guelph

TnC Isolation Protocol

Use BL21(DE3)Plyss competent cells.  Transform 0.5 uL of cTnC in a pet3a plasmid into the BL21(DE3)Plyss completent cells, plate them on LB plates containing Amp.  Put plates into 37 0C incubators.
 In the morning of next day, grow 1 colony in 3 ml of LB media containing AMP. In the evening of that same day, put 1 ml of cells into sterile 50 mL tubes containing 30 ml LB media with AMP (You can have 2,3 tubes), next morning:
1)  Grow in 1 L flasks of LB media, until OD is 0.6-0.8
2)  Add 0.4 mM IPTG
3)  Grow for 3 more hours
4)  Spin cells for 15 min at 8000 rpm.
5)  Resuspend cells in homogenation buffer (35 ml) 
6)  Sonicate cells output 6, duty cycle 60 %, 4 times 2 minutes.
7)  spin cells for 30 minutes, 11500 rpm.
8)  Slowly add 35% solid ammonium sulfate to the supernatant with constant stirring. 
9)  Put in a cold room for 1 hour, with constant stirring.
10)  Spin for 30 min at 11500 rpm.
11)  Add 5 mM Ca2+ to the supernatant, and load on phenylsepharose column, wash with buffer A (50 mM Tris, pH 7.5, 0.35 M ammonium sulfate, 0.5 mM CaCl2, 0.5 mM DTT) ~ 300 mL at room temperature.
12)  Elute with buffer B ( 50 mM Tris, pH 7.5, 0.15 M NaCl, 5 mM EDTA, 0.5 mM DTT).
13)  Collect 40 mL(in the 50 mL plastic tube), add 0.35 M ammonium sulfate, 15mM CaCl2, resuspend and reload on the phenylsepharose column.
14)  Wash with buffer A ~ 300 mL.
15)  Elute with buffer B.
16)  Collect 5 mL fractions.
17)  Determine OD @ 280 (Trp containing TnC’s).
*** If protein is low yield  ***
18)  For further purification, use fractions with OD280>0.2
19)  Dilute fractions with 50 mM Tris, pH 7.5, 0.5 mM DTT (1 part protein, 2 part Tris)
20)  Load on DEAE sepharose column.
21)  Wash with 50 mM Tris, pH 7.5, 0.5 mM DTT (~ 50-100 mL)
22a)  Elute with 50 mM Tris , 0.5 M NaCl, pH 7.5, 0.5 mM DTT collect 5 mL fractions.
22b)  Or use salt gradient to elute protein (0-1 M NaCl) and use a fraction collector.
23)       Determine OD @ 280.
24)       Dialyze against 3 changes of 4 L 10 mM MOPS, 90 mM KCL, 0.5 mM DTT, pH 7.0 at 4 0C.
 
Protein should be ready to go.
Homogenation buffer: 50 mM Tris, pH 7.5, 2 mM EDTA, 1 mM DTT, 1mM PMSF.