Protocols and Applications Guide (US Letter Size) - Promega
Protocols and Applications Guide (US Letter Size) - Promega
Protocols and Applications Guide (US Letter Size) - Promega
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||||| 5Protein Expression<br />
St<strong>and</strong>ard radioisotopic<br />
incorporation <strong>and</strong> detection<br />
M* M*<br />
Translation with<br />
incorporation of<br />
35S-met (1 hour)<br />
SDS PAGE (1 hour)<br />
Fix gel (30 minutes)<br />
Treat with enhancer<br />
(30 minutes)<br />
Dry gel (1 hour)<br />
Expose to<br />
X-ray film<br />
(4−10 hours)<br />
Transcend Biotinylated Lysine tRNA<br />
incorporation <strong>and</strong> detection<br />
L L<br />
Chemiluminescent<br />
detection<br />
Transfer to PVDF<br />
or nitrocellulose<br />
membrane (1 hour)<br />
L<br />
Block, bind<br />
Strep-HRP,<br />
wash (2 hours)<br />
Add<br />
Chemiluminescent<br />
Substrate <strong>and</strong><br />
expose to X-ray film<br />
(2−20 minutes)<br />
Translation with<br />
incorporation of<br />
biotinylated lysine (1 hour)<br />
SDS PAGE (1 hour)<br />
Colorimetric<br />
detection<br />
Transfer to PVDF<br />
or nitrocellulose<br />
membrane (1 hour)<br />
Block, bind<br />
Strep-AP,<br />
wash (2 hours)<br />
Add<br />
Western Blue ®<br />
Substrate to<br />
develop colored<br />
b<strong>and</strong>s (1−10 minutes)<br />
Time required = 8−14 hours Time required = 6 hours Time required = 6 hours<br />
Figure 5.8. Schematic of colorimetric <strong>and</strong> chemiluminescent detection of translation products.<br />
<strong>and</strong> background information about this system, please see<br />
Technical Bulletin #TB285 (www.promega.com<br />
/tbs/tb285/tb285.html).<br />
B. Translation Protocol<br />
• FluoroTect GreenLys in vitro Translation Labeling<br />
System (Cat.# L5001)<br />
• Nuclease-Free Water (Cat.# P1193)<br />
• translation system (e.g., TNT® Coupled<br />
Transcription/Translation System, Rabbit Reticulocyte<br />
Lysate, Wheat Germ Extract or E. coli S30 Extract)<br />
• complete amino acid mix or a combination of two minus<br />
amino acid mixes<br />
• salts, DTT <strong>and</strong> other components as needed to optimize<br />
the translation reaction<br />
• fluorescent imaging instrument (i.e., FluorImager® SI<br />
or FluorImager® 595 [Molecular Dynamics], both with<br />
a 488 argon laser; the Typhoon® 8600 [Molecular<br />
<strong>Protocols</strong> & <strong>Applications</strong> <strong>Guide</strong><br />
www.promega.com<br />
rev. 6/09<br />
Dynamics], with a 532nm excitation, or the FMBIO® II<br />
[Hitachi], with a 505 channel)<br />
0878MA08_3A<br />
PROTOCOLS & APPLICATIONS GUIDE 5-20
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