Protein truncation experiment

Diagram showing steps in a protein truncation test. First, a double stranded DNA helix is shown. The DNA region of interest is subjected to PCR amplification to ensure a sufficient number of copies. Next, in vitro transcription and translation occur. A strand of RNA is shown with a start codon on the 3' end. A protein, which is represented by a chain of red dots, is assembled during translation. The proteins are then analyzed using SDS-PAGE, a process that uses an electrical current in a gel matrix to separate proteins based on their molecular weight. A simple example of SDS-PAGE results is depicted, where bands in a gel tray represent the proteins. A full length protein band is shown near the loading well at the top of the tray, while a truncated protein band is shown as having travelled farther down the tray.

Figure 1. A schematic diagram showing the different steps of the protein truncation test.

The protein truncation test is composed of four major steps (Figure 1):

  1. Nucleic acid isolation; either genomic DNA, total RNA, or poly-A RNA.
  2. Amplification of a specific region of the gene of interest using PCR. In this step, a start codon (ATG) is added to the 5' end of the amplified DNA.
  3. In vitro transcription and translation of the product.
  4. Detection of the translated protein using polyacrylamide gel electrophoresis.