Biology Reference
In-Depth Information
24.2.1.2
Immunoaffinity and gel filtration chromatography
1.
SepSphere TM alprenolol agarose (CellMosaic, LLC, Worcester, MA)
2.
Nickel affinity gel (HIS-Select Nickel Affinity Gel, Sigma, St. Louis, MO)
3.
Centrifugal concentrators (100 kDa molecular weight cutoff)
4.
Gel filtration column—Sepharose 6 10/300 GL column (GE Life Sciences,
Piscataway, NJ)
5.
Carazolol
24.2.1.3
Crystallization
1.
Stock options Salt kit, HR2-245 (Hampton Research, Aliso Viejo, CA)
2.
Polyethylene glycol (PEG) 400
3.
1,4-Butanediol
4.
HR2-428 additive screen (Hampton Research, Aliso Viejo, CA)
5.
Monoolein (Nu-Chek Prep, Inc., Elysian, MN)
6.
Cholesterol
7.
LCP mixing devices (Emerald Biosystems, Bainbridge Island, WA)
8.
LCP sandwich screening plate (Swissci, Hampton Research, Aliso Viejo, CA)
9.
Cy3 mono-NHS-reactive dye (GE Life Sciences)
24.2.2
Methods
24.2.2.1
Protein purification
24.2.2.1.1
Crude membrane preparations
Prepare insect cell pellet (from 3 L of cell culture) expressing the
2-AR(E122W)/
T4L construct. All following steps should be performed on ice or at 4
C. The pu-
rification of
b
2-AR is designed to be completed in 2-3 days with an extra day ded-
icated to crystallization.
Centrifuge the Sf9 cells in phosphate saline buffer (PBS) at 50,000
b
g
for 15 min
and homogenize the resulting cell pellet in
300 mL of minimal buffer (10 mM
HEPES, pH 7.5, 1 mM MgCl
2
, 2 mM KCl and one protease inhibitor tablet per
50 mL) with 20 up and down strokes in a Dounce homogenizer. Centrifuge the result-
ing homogenate at
g
for 30 min and discard the supernatant. Repeat the
above process at least three times.
Homogenize the combined pellet from the last step into
50,000
400 mL of minimal
buffer supplemented with 1 M NaCl and Benzonase. Repeat the membrane washing
by centrifugation as in the previous step until a tight pellet is obtained.
24.2.2.1.2
Solubilization
Homogenize the washed membranes in a final volume of 500 mL with solubilization
buffer (50 mMHEPES, pH 7.5, 0.15 MNaCl, 1 mMMgCl
2
, 2 mMKCl, and protease
inhibitor cocktail). Then, add PNGase F and more Benzonase. Homogenize with 20 up
and down strokes in Dounce homogenizer, add iodoacetamide (2 mg/mL), and incu-
bate at 4
C for 30-45 min. Add DDM and CHS from a 10
stock to final concen-
trations of 0.5% and 0.1%, respectively, and rotate the homogenate for at least 6 h.
