Biology Reference
In-Depth Information
Table 3.1 List and composition of the buffers used for DCX purification
Buffer (pH 7.2)
Composition
1
Lysis—low salt
40 mM HEPES
50 mM NaCl
50 mM Imidazole
10% Glycerol
1% Triton X-100
10 mM b-Mercaptoethanol
0.5 mM EDTA
1 mM PMSF
One protease inhibitor tablet
0.015 mg/mL Aprotinin, leupeptin, pepstatin
0.15 mg/mL DNase I
2
Lysis—high salt
25 mM HEPES
1 M NaCl
50 mM Imidazole
10 mM b-Mercaptoethanol
0.5 mM EDTA
1 mM PMSF
3
50 mM Imidazole Ni-NTA wash
25 mM HEPES
300 mM NaCl
50 mM Imidazole
10 mM b-Mercaptoethanol
0.5 mM EDTA
1 mM PMSF
4
250 mM Imidazole Ni-NTA elution
25 mM HEPES
300 mM NaCl
250 mM Imidazole
0.5 mM EDTA
10 mM b-Mercaptoethanol
5
Gel filtration
25 mM HEPES
300 mM NaCl
0.5 mM EDTA
2 mM DTT
Finally, the His-tagged DCX is cleaved using recombinant tobacco etch virus
protease (rTEV; gift from EMBL Heidelberg). Complete cleavage is achieved by
adding 1% w/w rTEV (protease/fusion protein) and incubating at 4 C overnight
( Fig. 3.3 D). A second Ni-NTA column (300 m L resin) retains uncleaved His-DCX
as well as the rTEV; addition of 20 mM imidazole in the column buffer reduces
unspecific interaction of the cleaved protein with the resin. In the original protocol,
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