24 Protein Techniques
369
The reaction is incubated at 30°C for 60 min. One should always include a
negative control containing no added mRNA. Basedon the synthesis of globin the reticulocyte lysate system translates about 80 amino acids per min.
Therefore, a 100-kDa protein would require about 10-15 min to synthesize.
Protease inhibitors can be added to the reaction although the choice is not
broad. Soybean trypsin inhibitor (I mg/ml) and ovomucoid (0.5 mg/ml) can
be used safely. EGTA (2.7 mM) and phenylmethylsulfonylfluoride (PMSF;
l7J.lg/ml) can also probably be used. It should be noted that different lots of
methionine may contain an inhibitor of translation. Therefore, it is best to
use either translation-grade methionine from New England Nuclear or
Translabel from ICN. The New England Nuclear product has a higher specific activity (800-1200 Ci/mmol) than Translabel (800 Ci/mmol), but Translabel also contains radiolabeled cysteine. Methionine is very sensitive to
oxidation and should be storedas single-use aliquots at -70°C.
Reaction products are then analyzed by TCA precipitation to determine
the number of counts incorporated. Under ideal conditions one should incorporate 3-5 x 10 6 counts (0.5-l.5J.1Ci) of3 5 S-methionine per reaction mixture. A small aliquot ofthe reaction (5J.1l) is added to 20 J.ll of sample buffer,
heated, and the products ofthe reaction are separated on an SDS-polyacrylamide gel. Potential artifacts include labeled peptidyl-tRNA resulting from
the translation of small fragments of globin mRNA (6). This migrates as a
group of bands at 25-30 kDa. Their labeling is suppressed by inhibitors of
protein synthesis and by the addition of efficiently translated mRN As to the
assay. As a result these fragments are most troublesome when one is trying
to translate poorly initiating or scarce messages. Fortunately, these fragments can easily be eliminated if the sample is treated with 50-100 J.lg/
ml ofRNase A for 10 min at 30°C. Another artifactual polypeptide migrating
at 42-45 kDa may also be seen (6). It is thought to result from a tRNA-dependent but ribosome-independent addition of methionine to a lysate protein. If this polpeptide is a major problern its mobility can be altered by
omitting the customary heating step before loading the gel. It will then migrate at 220 kDa. Omitting the heating step has little effect on the mobility of
the translation products. Using 35 S-methionine, it should be possible to detect 100-200 nglml of protein in 1-2 hrs employing fluorography. The Iimit
of detection of this method is determined by the small amount oflysate that
can be loaded in each lane (2-5 J.ll), since the lysate itself has a very high
protein concentration (50-60 mg/ml). In comparison, immunoprecipitation can detect the translation of as little as 5-20 ng/ml of protein.
369
The reaction is incubated at 30°C for 60 min. One should always include a
negative control containing no added mRNA. Basedon the synthesis of globin the reticulocyte lysate system translates about 80 amino acids per min.
Therefore, a 100-kDa protein would require about 10-15 min to synthesize.
Protease inhibitors can be added to the reaction although the choice is not
broad. Soybean trypsin inhibitor (I mg/ml) and ovomucoid (0.5 mg/ml) can
be used safely. EGTA (2.7 mM) and phenylmethylsulfonylfluoride (PMSF;
l7J.lg/ml) can also probably be used. It should be noted that different lots of
methionine may contain an inhibitor of translation. Therefore, it is best to
use either translation-grade methionine from New England Nuclear or
Translabel from ICN. The New England Nuclear product has a higher specific activity (800-1200 Ci/mmol) than Translabel (800 Ci/mmol), but Translabel also contains radiolabeled cysteine. Methionine is very sensitive to
oxidation and should be storedas single-use aliquots at -70°C.
Reaction products are then analyzed by TCA precipitation to determine
the number of counts incorporated. Under ideal conditions one should incorporate 3-5 x 10 6 counts (0.5-l.5J.1Ci) of3 5 S-methionine per reaction mixture. A small aliquot ofthe reaction (5J.1l) is added to 20 J.ll of sample buffer,
heated, and the products ofthe reaction are separated on an SDS-polyacrylamide gel. Potential artifacts include labeled peptidyl-tRNA resulting from
the translation of small fragments of globin mRNA (6). This migrates as a
group of bands at 25-30 kDa. Their labeling is suppressed by inhibitors of
protein synthesis and by the addition of efficiently translated mRN As to the
assay. As a result these fragments are most troublesome when one is trying
to translate poorly initiating or scarce messages. Fortunately, these fragments can easily be eliminated if the sample is treated with 50-100 J.lg/
ml ofRNase A for 10 min at 30°C. Another artifactual polypeptide migrating
at 42-45 kDa may also be seen (6). It is thought to result from a tRNA-dependent but ribosome-independent addition of methionine to a lysate protein. If this polpeptide is a major problern its mobility can be altered by
omitting the customary heating step before loading the gel. It will then migrate at 220 kDa. Omitting the heating step has little effect on the mobility of
the translation products. Using 35 S-methionine, it should be possible to detect 100-200 nglml of protein in 1-2 hrs employing fluorography. The Iimit
of detection of this method is determined by the small amount oflysate that
can be loaded in each lane (2-5 J.ll), since the lysate itself has a very high
protein concentration (50-60 mg/ml). In comparison, immunoprecipitation can detect the translation of as little as 5-20 ng/ml of protein.