Biomedical Engineering Reference
In-Depth Information
11. These Arf1-containing fractions are concentrated by ultrafi ltra-
tion (Amicon Ultra fi lter) to approximately 2 mL.
12. The sample is fractionated at a fl ow rate of 1 mL/min on the
Superdex 75 column equilibrated with Superdex running buf-
fer. Fractions of 3 mL are collected.
13. Fractions 51-80 are tested for the presence of Arf1 and con-
taminating proteins by analyzing 25-
L samples on parallel
15 % SDS-gels by immunoblotting and Coomassie Blue stain-
ing, respectively.
14. Arf1-containing fractions (typically fractions 60-70) are pooled
(
see
Note 9
) and concentrated to 1 mg/mL by ultrafi ltration.
15. The purifi ed protein is aliquoted, shock-frozen in liquid nitro-
gen, and stored at −80 °C. Approximately 0.5 mg Arf1 could
be typically purifi ed from a 2.5 L culture.
ʼ
His
6
-tagged wild-type and mutant amphiphysins are expressed in
bacteria and isolated in a one-step purifi cation by nickel chelate
chromatography adapted from the provider's protocols.
3.4 Expression
and Purifi cation
of Amphiphysin
1. Twenty milliliters of an overnight culture of
Escherichia coli
BL21 with plasmid pET24d-Amph1 or pET24d-Amph2 in
LB/Kana is used to inoculate 2 L LB/Kana. The culture is
grown at 37 °C for 3-4 h until OD
600
reaches 0.7.
2. Expression is induced by addition of 0.5 mM IPTG and fur-
ther culturing for 6 h at 30 °C for amphiphysin 1, and with
1 mM IPTG and further culturing for 4 h at 30 °C for amphi-
physin 2 (
see
Note 10
).
3. The cells are harvested by centrifugation at 5,000 ×
g
for
15 min (Sorvall GS3 rotor, 5,400 rpm).
4. The pellets are resuspended in an equal volume of PBS, trans-
ferred into a 50-mL Falcon tube, pelleted again, and stored
frozen at −80 °C.
5. The pellet is resuspended in 20 mL sonication buffer supple-
mented with 2× protease inhibitor cocktail and 2× PMSF.
6. Sonicate eight times for 15 s at an amplitude of 25 % in 1-min
intervals with the tube immersed in ice water.
7. The material is centrifuged for 1 h at 100,000 ×
g
(Thermo
Scientifi c T-1270 rotor, 40,000 rpm).
8. The supernatant is loaded twice at a fl ow rate of 1 mL/min
onto the 1-mL HisTrapp FF column equilibrated in sonication
buffer.
9. The column is washed and eluted at 1 mL/min with sonica-
tion buffer containing increasing concentrations of imidazole:
20 mL with 50 mM, 5 mL with 75 mM, and 2 mL each of
