Biology Reference
In-Depth Information
2.
The digitonin concentration has been optimized for permeabilization of
HT1080 cells (i.e., the lowest concentration of digitonin that results in 100%
permeabilization). If a different cell line is used, the concentration of digitonin
required for permeabilization should be assessed by titration. It is not essential to
trypan blue stain each batch of SP cells, although this is recommended if the proce-
dure is not used routinely.
3.
The yield of mRNA can be increased by a further addition of RNA polymerase
(1
µ
L) after 1 h.
4.
To minimize degradation of the mRNA, the use of sterile pipet tips, microcentrifuge
tubes is recommended. If the yield is low or the RNA is partially degraded it is
possible that apparatus or solutions have been contaminated with RNases.
5.
The authors recommend that the translation protocol is optimized for each different
mRNA transcript as the optimal salt concentration (KCl and MgOAc) may vary.
6.
To test the translation efficiency of a new RNA preparation, a single 25-
µ
L reac-
tion including 4
µ
L of sterile water instead of SP cells can be prepared.
7.
If there are no protein bands then the RNA may need to be heated to 60
C for
10 min prior to translation in order to denature any secondary structure. Additional
products with molecular weights smaller than the major translation product may be
observed because of ribosome binding to “false” start sites downstream of the
initiation codon.
°
8.
The translocated and nontranslocated forms of the protein usually migrate differ-
ently on a reducing SDS-PAGE gel owing to modification of the nascent chain in
the ER lumen. The translation products not treated with proteinase K will contain a
mixture of these forms, the ratio of which is dependent upon the efficiency of trans-
location. As the nontranslocated polypeptides are selectively degraded by addition
of proteinase K, it is possible to assess which translation product corresponds to
each form. The nontranslocated form will also comigrate with the polypeptide syn-
thesized in the absence of SP cells.
In the case of transmembrane proteins, treatment with proteinase K results in an
increase in electrophoretic mobility corresponding to loss of the cytoplasmic
domain that is accessible to the enzyme. The translocated polypeptide may migrate
faster than the nontranslocated form resulting from signal peptide cleavage that
occurs when the nascent chain enters the ER lumen. However, this may only
be detected if no other covalent modification of the polypeptide occurs. Usually,
the translocated polypeptides will exhibit decreased electrophoretic mobility
because of glycosylation. In the case of procollagens, hydroxylation of proline and
lysine residues also results in decreased electrophoretic mobility.
9.
The reduced and nonreduced samples should be separated by a gap of two lanes in
order to prevent reduction of the nonreduced samples by DTT that may diffuse
across the gel matrix during electrophoresis.
References
1. Wilson, R., Oliver, J., Brookman, J. L., High, S., and Bulleid, N. J. (1995) Devel-
opment of a semi-permeabilised cell system to study the translocation, folding,
assembly and transport of secretory proteins.
Biochem. J.
307,
679-687.
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