Biology Reference
In-Depth Information
4.
Sf900II-serum free medium for insect cell culture (Gibco-BRL, Gaithersburg,
MD, 10902-088) (
see
Note 5
).
5.
Fetal bovine serum (Gemini Bio-Products; 100 - 106), heat-inactivated sterile-
filtered, stored at -20
°
C (
see
Note 6
).
6.
Gentamycin sulfate (Gibco-BRL; 15710-015).
7.
Amphotericin B (Fungizone, Gibco-BRL; 15295-017).
8.
Tissue culture plastic: 25cm
2
-, 75cm
2
-vented caps, 96-well plates (Corning, NY).
9.
Magnetic spinner flasks: 50 mL, 250 mL, 500 mL, and 1 L (Bellco Glass, Inc).
10.
Multi-channel magnetic stir plate (Bellco; Multi-Stir 9 model) (
see
Note 7
).
11.
Incubator stringently maintained at 27
°
C.
12.
Protease inhibitor stock solutions: 1
M
EDTA, pH 8.6 in dd water; 150 m
M
PMSF
in isopropanol.
13.
Dialysis tubing (Spectrum; 132703).
14.
Elution gradient mixer (>200 mL per chamber) with magnetic stir bar apparatus.
15.
Conventional heparin-sepharose CL-6B column (Pharmacia, Uppsala, Sweden),
bed volume >100 mL, equipped with UV monitor and fraction collector.
16.
Conventional DEAE-sepharose CL-6B column (Sigma, St. Louis, MO, DCL-6B-
100 CL-6B), bed volume >25 mL, equipped with UV monitor.
17.
Aquacide (Calbiochem; 17851) (
see
Note 8
).
18.
Programmable HPLC apparatus: TSK-Gel Heparin-5PW (Toso-Haas; 14444)
column, dimensions (7.5 cm
×
8 mm inner diameter).
19.
Buffers:
a. 50 m
M
NH
4
HCO
3
pH 8.0, 5 m
M
EDTA, 1 m
M
PMSF.
b. with 1
M
NaCl.
c. 150 m
M
Tris HCl pH 7.4, 1 m
M
EDTA, 0.1 m
M
PMSF.
d. (c) with 1
M
NaCl.
e. Tris Buffered Saline (TBS) pH 7.4, 0.02% NaN
3
.
3. Methods
3.1. Cloning and Transfection
1.
Clone the cDNA encoding G domain downstream of the promoter and appropri-
ate signal sequence using standard molecular cloning protocols (
see
Table 1
).
Seed a 25-cm
2
10
6
Sf9 cells in log phase and allow the cells to
attach for at least 30 min (
see
Note 9
).
2.
flask with 1
×
3.
Aspirate SF900II medium and replace with 1.5 mL BaculoGold Transfection
Buffer A (supplied in the BaculoGold Transfection Kit) making sure the entire
dish surface is covered.
4.
In a sterile 5 mL-polycarbonate tube, combine 5
µ
g recombinant plasmid with
0.5
µ
g BaculoGold baculovirus DNA. Incubate 15 min at room temperature.
5.
Add 1.5 mL BaculoGold Transfection Buffer B to the DNA mixture and mix by
inversion.
Add dropwise with agitation
to Sf9 cells in Buffer A (
see
Note 10
).
Allow the transfection to continue for 4 h at 27
°
C.
6.
Aspirate transfection medium and replace with 5 mL fresh SF900II medium.
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