Protein Truncation Test
Figure 1. A schematic diagram showing the different steps of the protein truncation test. 
A protein truncation test is a powerful method to evaluate DNA mutations that result in protein truncation in vitro. Based on the impact to the translated proteins, DNA mutations are classified into frameshift mutation, nonsense mutation, missense (nonsynonymous) mutation, neutral mutation, and silent mutation. Nonsense mutation results in a premature stop codon. This means that the resulting protein will be terminated earlier than what it is supposed to be and can be detected using a protein truncation test. This shorter protein is also called truncated protein. With a protein truncation test, we do not need to use an animal as a model system to synthesize the protein; instead, we have an in vitro system where the resulting protein can be synthesized without the requirement of living cells.
Protein truncation tests are often used to analyze disease-related mutations, for example, in cancer where the mutation results in a nonfunctional protein. From protein truncation tests, we can conclude whether there is a mutation in a particular part of the gene that results in protein truncation; however, we do not have information regarding where exactly the mutation is located and what is the resulting DNA sequence (for example, it could be a substitution creating a stop codon or a deletion resulting in a frameshift and then a stop codon or others). To find out more information regarding the mutation, we need to perform DNA sequencing as a validation method. With DNA sequencing, we will be able to know the exact sequence of DNA that results in a truncated protein.
The protein truncation test is composed of four major steps (Figure 1):
- Nucleic acid isolation; either genomic DNA, total RNA, or poly-A RNA.
- 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.
- In vitro transcription and translation of the product.
- Detection of the translated protein using polyacrylamide gel electrophoresis.
Polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis is a technique used to analyze proteins on the basis of their size. It is quite similar to agarose gel electrophoresis that is used to analyze DNA after performing the PCR. Unlike DNA, proteins can be folded into complex 3D structures. Before running them on the gel, we must first denature this 3D structure of the proteins and make them linear. We can do this by adding sodium dodecyl sulfate (SDS), which is a detergent that unravels proteins to make them linear. SDS is also required to charge the proteins negatively.
When the proteins are linearized and have a negative charge, we can load them onto a polyacrylamide gel and apply electrical charge. The negatively charged proteins will travel through the porous polyacrylamide toward the positive pole, which is located at the end of the gel. The longer proteins will travel slower than the shorter proteins; this is how we can separate proteins on the basis of their length using polyacrylamide gel electrophoresis. In protein truncation test, the wild-type proteins will migrate less than the truncated proteins that are shorter.