Biomedical Engineering Reference
In-Depth Information
B 0mM NaHCO
3
,5mM imidazole, 0.2 mM DTT or DTE,
0.1 mM PMSF, pH 6.8
C . 5M sucrose, 5 mM imidazole, pH 7.2
D .6M KCl, 10 mM imidazole, pH 7.2
D
18% sucrose (w/v) in Soln. D
D
27% sucrose (w/v) in Soln. D
All steps have to be done in a cold chamber or in an ice bath.
Crush 250 g of deeply frozen porcine left ventricle and thaw in
900 ml of Soln. A. Homogenize the tissue using a Warring blender
(low speed) for 20 s. Spin the homogenate in a Sorval GS-3 rotor or
Beckman-Coulter JA-10 rotor at 3000
g (4500 rpm)for15min.
Combine the pellets, suspend in 750 ml of Soln. A and homog-
enize again for 20 s. Repeat centrifugation. Divide the pellet into 12
portions, fill up each portion with Soln. B to 120 ml,andhomoge-
nize each portion using a Polytron homogenizer at 60% of maximal
rpm, three times for 15 s each.
Combine the homogenates and spin at 11 000
×
g (Sorval GS-3
rotor or Beckman Coulter JA-10 rotor: 8500 rpm)for20min.
Centrifuge the combined supernatants at 57 000
×
g (Beckman
Coulter Type 45Ti rotor: 26 000 rpm)for40min; discard the super-
natants.
Suspend the pellets (crude membrane fraction) using a glass-
Teflon homogenizer in Soln. D to a final volume of 24 ml.
Prepare continuous density gradients from Soln. D
and Soln.
D
, cover with the suspension of the crude membrane suspension
and centrifuge with 60 000
×
g
max
,respectively,for
90 min (e.g., Beckman Coulter SW 28 rotor: gradient from 16.5 ml
Soln. D
and 16.5 ml Soln. D
per tube; 24 000 rpm).
Carefully suck off the white zone located about 1 cm below the
meniscus, combine the material from all tubes and dilute 1:2 with
Soln. D. Spin at 60 000
×
g
av
and 83 000
×
g
max
for 45 min (e.g., Beckman Coulter
Type 50.2Ti rotor: 40 000 rpm).
The resulting pellets are highly purified sarcolemmal mem-
brane vesicles. Suspend the pellets in a small volume of Soln. D,
aliquote, and freeze in alcohol-carbon dioxide or liquid nitrogen.
Store at −70
◦
C.
To characterize the preparation, determine marker enzymes,
e.g., p-nitrophenyl phosphatase (PNPase), ouabain-sensitive Na,K-
dependent ATPase, or dihydropyridine receptor complex (L-type
voltage dependent calcium channel).
×
References
Vetter R, Haase H, Will H (1982) FEBS Lett 148:326
To identify the cellular organelles, enzymes associated with well-
established functions are used. The enrichment of an organelle
