Target Gene Amplification by PCR/RT-PCR with Specialized Epitope Primers
Radioactive handling Cell-FreeTranslation with required Radiolabeled Amino Acids
Cell-Free Translation with Engineered tRNAs
Low throughput applications only
High throughput ELISA-based PTT screening (>1000 samples)
8-48 hrs for detection
Not amenable to automation
Chemiluminescence Detection 5 min for detection
Total Assay Time: 10-50 hrs
Automatic Readout t
Follow-up Analysis t
PAGE-based PTT t
Positive samples can be validated and truncation location identified
Amenable to automation
Total Assay Time: 1-2 hrs
Fig. 1 Various steps involved in typical protein truncation test. (View this art in color at www.dekker.com.)
In addition, several attempts have been made to scan entire genes for truncating mutations using a combination of genomic DNA and mRNA (Table 1). In the case of RNA-based PTT, mRNA is first reverse transcribed to cDNA, which then functions as a template for amplification. The best source of RNA is cells in which the target gene is abundantly expressed. However, for practical reasons RNA is generally isolated from freshly drawn peripheral blood lymphocytes.
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The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.