Semipermeabilized Cells to Study Procollagen Assembly - Methods in Molecular Biology: Extracellular Matrix Protocols - page 4

10. 1 mg/mL micrococcal nuclease in sterile water, stored at -20 ° C.

11. 0.4 M EGTA (stored at -20 ° C).

2.2. Transcription In Vitro
1. 10 g linearized plasmid DNA, containing gene of interest downstream form a

viral polymerase promoter, in RNase-free water.

µ

2. 5X transcription buffer (400 m M HEPES buffer, pH 7.4, 60 m M MgCl 2 , 10 m M

Spermidine).

3. Nucleotide triphosphates (ATP, UTP, CTP, and GTP) (25 m M each) (Boehringer

Mannheim, Mannheim, Germany).

4. 100 m M DTT (Sigma).

5. T3/T7 RNA polymerase (50 U/ µ L) (Promega, Madison, WI).

6. RNase inhibitor (Promega).

2.3. Translation In Vitro
1.

Flexi™ rabbit reticulocyte lysate.

2. Amino acid mix (minus methionine).

3. 2.5 M KCl.

4. EasyTag™ 35 S-methionine.

All reagents were supplied by Promega and stored at -70 ° C, except the

35 S-methionine that is supplied by NEN Dupont and stored at 4

° C.

2.4. Proteinase K Treatment
1. 2.5 mg/mL proteinase K in sterile H 2 O.

2. 0.1 M CaCl 2 .

3. 10% Triton X-100 stored at 4 ° C.

4. 0.1 M PMSF in isopropanol.

5. All reagents stored at -20 ° C.

3. Methods
3.1. Preparation of SP Cells
This procedure uses a modified protocol based on that of Plutner et al. (2) ,
which has been adapted for the cell-free expression of proteins (1) . Treatment
of mammalian cells with a low concentration of the detergent digitonin renders
the plasma membrane permeable to the components of the cell-free translation
system whereas retaining the ER membrane in a functionally intact state. This
selective permeabilization of the plasma membrane is a consequence of the
cholesterol-binding properties of digitonin. As cholesterol is only a minor
constituent of the internal membrane system of the cell, the ER and Golgi
networks remain intact, although some swelling of the ER is observed ( see
Note 1 ).