Peter Novick Lab, Department of Cell Biology Yale University School of Medicine
http://info.med.yale.edu/cellbio/Novick/second/protocols/sucrose.pdf
Cells (200 OD599 Units) were grown at 25oC in YP medium containing 2%
glucose. The cells were pelleted and transferred to YP supplemented with
0.2% glucose and incubated with shaking for 1 hour at 37oC. After
washing once with 10 mM NaN3, the cells were resuspended in spheroplast
media (50 mM Tris pH7.5, 10 mM NaN3, 1.4 M sorbitol, 40 mM B-
mercaptoethanol, 0.125 mg/ml Zymolase-100T) and incubated at 37oC for
45 min. Spheroplasts were pelleted, cooled on ice, and resuspended in 20
ml ice cold lysis buffer (0.8 M sorbitol in 20 mM Triethanolamine, 1 mM
EDTA pH 7.2) containing 1 mM PMSF and 10 ul/10 ml protease inhibitor
cocktail: leupeptin, chymostatin, pepstatin, aprotinin, and antiparin 1
mg/ml. All following steps were performed at 4oC. The suspension was
homogenized 20 times in a 40 ml Wheaton tissue grinder, pestle A, and
centrifuged at 450 xg for 3 min. The pellet (P1) was resuspended in the
same volume lysis buffer, homogenized and centrufuged as above, and the
supernatants (S1) pooled. 2 ml of 1 M MES, 2-(N-morpholino) ethane
sulfonic acid, pH 6.5 was added to S1 and the supernatant was spun at
10,000 xg for 10 min. The supernatant (S2) was spun at 100,000 xg for 1
hour to produce a P3 pellet and S3 supernatant. The P2 and P3 pellets
were resuspended in 2 ml of 55% sucrose (wt/wt) containing 10 mM MES
pH 6.5 and homogenized 4 strokes in a 2 ml Wheaton tissue grinder. The
P2 or P3 homogenate was placed at the bottom of a SW41 tube and
overlayed with the following sucrose solutions: 1 ml 50%, 1 ml 47.5%, 1.5
ml 45%, 1.5 ml 42%, 1.5 ml 40%, 1 ml 37.5%, 1 ml 35%, 1 ml 30%, all
containing 10 mM MES pH 6.5. The gradients were spun at 170,000 xg in a
SW41 rotor (Beckman) for 16 hrs. Fractions were collected from the
bottom and any pellet resuspended in an identical volume (as other
fractions) of 55% sucrose and labelled as fraction 1.
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