Biology Reference
In-Depth Information
4.
Gradient solutions, with densities to be adjusted with two hydrometers
(ranges 1.060-1.130 and 1.120-1.190 g/mL)
1.10 g/mL (
107 mL of 45% sucrose
þ
93 mL Kuhn's buffer)
1.13 g/mL (
167 mL of 45% sucrose
þ
72 mL Kuhn's buffer)
41 mL Kuhn's buffer)
5.
40-50 mL high-speed, transparent centrifuge tubes
6.
Swinging-bucket rotor able to reach 26,500
1.15 g/mL (
205 mL of 45% sucrose
þ
g
7.
10 mL syringes with luer locks and Popper Laboratory Pipetting Needles,
14G
6 in.
8.
Funnel
9.
Gauze sponges, 12 ply, 4
4 in.
24.1.1.2
Nonyl-glucoside/Zn(OAc)
2
extraction of rhodopsin
1.
0.5 M 2-(
N
-morpholino)ethanesulfonic acid (MES), pH 6.3-6.4
2.
1 M Zn(OAc)
2
3.
10% nonyl-
b
-
D
-glucoside (NG)
4.
UV buffer: 1-5 mM dodecyl-
b
-
D
-maltoside (DDM), 50 mM Tris, pH 7.4,
100-150 mM NaCl, and 1 mM hydroxylamine
24.1.1.3
Immunoaffinity purification
1.
1D4 monoclonal antibody coupled to CNBr-activated Sepharose (GE Healthcare
Life Sciences) or agarose (Pierce) (90-95 mL of settled gel)
2.
Glass column (1-2.5 cm diameter
20-50 cm length)
3.
Peristaltic pump
4.
Fraction collector
5.
Washing buffer: 25-50 mM NG in 150 mM Tris, pH 7.4, 280 mM NaCl, and
6 mM KCl
6.
Elution buffer: 0.5-1 mg/mL TETSQVAPA peptide in washing buffer
24.1.1.4
(NH
4
)
2
SO
4
-induced phase separation
1.
Solid (NH
4
)
2
SO
4
2.
40-50 mL high-speed, transparent centrifuge tubes
3.
Glass rod and small magnetic rod
4.
0.5 M MES, pH 6.3-6.4
5.
2 mL, dolphin-nose bottom, microfuge tubes
24.1.1.5
Crystallization
1.
24-well, greased crystallization plates
2.
Transparent microbridges
3.
Basic stereo microscope, with 30
and 60
magnification
4.
Thick cover slides (0.96 mm
22 mm)
5.
Crystallization buffer: 0-110 mM NG in 50-100 mM MES, pH 6.3-6.4, 12 mM
b
-mercaptoethanol, 0.1% NaN
3
, and 2.5-5% MERPOL HCS
6.
Reservoir buffer: 3-3.4 M (NH
4
)
2
SO
4
in 10-50 mM MES, pH 6.3-6.4
22 mm
