Semipermeabilized Cells to Study Procollagen Assembly - Methods in Molecular Biology: Extracellular Matrix Protocols - page 7

3. Incubate the three samples on ice for 20 min, followed by a further incubation of

the samples on ice for 5 min with 1 m M PMSF to inhibit the proteinase K.

4. Prepare the samples for electrophoresis by adding 5 µ L of each reaction to 15 µ L

of SDS-PAGE buffer containing 2 µ L DTT (1 M ).

5. The samples should be separated through an 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 8 ).

3.5. Analysis of Disulfide Bond Formation
The native conformation of proteins entering the secretory pathway is often
stabilized by disulfide bonds. The formation of intrachain disulfide bonds in a
particular domain of a nascent polypeptide may represent a key event in the
folding pathway. In the case of fibrillar procollagen chains, formation of
the correct intrachain disulfide bonds in the carboxy-terminal domain (the
C-propeptide) is necessary for the folding of these domains and is a prerequi-
site for trimer formation (10 , 11) . The trimers in turn are stabilized by interchain
disulfides. The formation of disulfide bonds during folding can be moni-
tored over time by trapping folding intermediates using the alkylating
reagent N-ethyl maleimide (NEM) (12) . The formation of intrachain disul-
fide bonds generally increases the electrophoretic mobility of proteins during
SDS-PAGE analysis, provided the sample is analyzed under nonreducing
conditions. In contrast, multisubunit proteins that are stabilized by interchain
disulfide bonds have a faster migration when the protein is treated with reduc-
ing agents that cause dissociation into constituent monomers.
1. Prepare a 100- µ L translation mix and divide this into four aliquots of 25 µ L in

separate microcentrifuge tubes

2. At intervals of 15 min, remove one of the tubes and add NEM to a final concen-

tration of 20 m M and place on ice for the remainder of the time-course.

3. Isolate and “wash” the SP cells as described in steps 1 and 2 ( Subheading 3.5. ).

4. Solubilize each of the washed cell pellets in 50 µ L SDS-PAGE buffer and then

transfer 25 µ L of each sample into fresh tubes containing 2 µ L DTT. Boil the

samples for 5 min prior to electrophoresis ( see Note 9 ).

4. Notes
1. The procedure takes approximately 1 h and should be carried out immediately

prior to using the SP cells for translation in vitro, SP cells do not efficiently
reconstitute the translocation of proteins after storage. It is also advisable to use a
minimum of one 75 cm 2 flask of cells (75-90% confluent) as it is difficult to
work with a smaller quantity of cells. The size of the cell pellet will usually
decrease during the procedure because of loss of the cell cytosol that is accompa-
nied by a decrease in cell volume.