Semipermeabilized Cells to Study Procollagen Assembly - Methods in Molecular Biology: Extracellular Matrix Protocols - page 6

2. Add 3 µ L of the appropriate RNA polymerase (150 U) and incubate at 37 ° C for 2 h

( see Note 3 ).

3. The RNA can be extracted with phenol/chloroform 1:1, then twice with chloro-

form and precipitate by adding NaOAc, pH 5.2 to a final concentration of
300 m M and 3 vol of ethanol. The RNA pellet is resuspended in 100 µ L RNase-free

H 2 O containing 1 m M DTT and 1 µ L RNasin.

4. To assess the yield of RNA, a 1- µ L aliquot should be removed and analyzed on a

1% agarose gel ( see Note 4 ).

3.3. Translation In Vitro
The translation of proteins in vitro can be performed using either wheat germ
extracts or rabbit reticulocyte lysates that contain ribosomes, tRNAs, and a
creatine phosphate-based energy regeneration system.
1. Prepare a 25- µ L reaction mixture containing 17.5 µ L Flexi™ lysate, 0.5 µ L amino

L EasyTag 35 S-methionine, 1 acids, 0.5 µ L KCl, 1.5 µ µ L mRNA, and 4 µ L SP

cells ( see Notes 5 and 6 ). Incubate the translation sample at 30 ° C for 60 min and

then place on ice.

2. Prepare the translation sample for SDS-PAGE by adding 2 µ L of the product to

15 L SDS-PAGE sample buffer (0.0625 M Tris/HCl pH 6.8, SDS (2% w/v),

glycerol (10% v/v), and bromophenol blue) plus 2

µ

µ L DTT (1 M ) and boiling the

sample for 5 min.

3. The samples should be separated through a SDS-PAGE gel appropriate for the

expected molecular weight for the protein of interest. After running, the gel
should be dried and exposed to autoradiography film ( see Note 7 ).

3.4. Proteinase K Protection
A “protease protection” assay is a method used to determine whether the
nascent chains are targeted to the ER membrane and translocated into the ER
lumen of the SP cells. The translation samples are treated posttranslationally
with proteinase K, which rapidly digests any nontranslocated translation prod-
ucts, whereas fully translocated proteins are protected by the lipid bilayer of
the ER membrane. A control sample is incubated in the presence of proteinase
K and Triton X-100, which solubilizes the ER membrane, and, therefore ren-
ders the translocated protein susceptible to proteinase K digestion ( see also
Note 8 ).
1. Prepare a 25- L translation reaction including freshly prepared SP cells as

described in Subheading 3.3. After translation, place the sample on ice and gen-
tly disperse the SP cells using a pipet tip.

µ

2. Divide the translation mixture into three microcentrifuge tubes containing 3 L

aliquots. One sample is used as a nontreated control. To the other two tubes, add
1

× 8- µ

L Proteinase K (2.5 mg/mL). To one of these
samples, also add Triton X-100 to a concentration of 1% (v/v).

µ L CaCl 2 (100 m M ) and 1 µ