Basic Techniques in Protein Analysis


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In two-hybrid screening, the binding domain of a transcription factor is separated from the activator domain. A bait protein is attached to the D N A binding domain of a transcription factor, and a prey protein is attached to the activator domain. If the prey catches the bait, in other words, binds to it, transcription of a reporter gene occurs. If the prey does not catch the bait, no transcription occurs. Scientists use this transcriptional activation to determine if interaction between the bait and prey has occurred.
Scientists use two-hybrid screening to determine whether two proteins interact. In this method, a transcription factor splits into a DNA-binding domain (BD) and an activator domain (AD). The binding domain is able to bind the promoter in the activator domain’s absence, but it does not turn on transcription. The bait protein attaches to the BD, and the prey protein attaches to the AD. Transcription occurs only if the prey “catches” the bait. Source: OpenStax Biology 2e

OpenStax Biology 2e

The ultimate goal of proteomics is to identify or compare the proteins expressed from a given genome under specific conditions, study the interactions between the proteins, and use the information to predict cell behavior or develop drug targets. Just as scientists analyze the genome using the basic DNA sequencing technique, proteomics requires techniques for protein analysis. The basic technique for protein analysis, analogous to DNA sequencing, is mass spectrometry. Mass spectrometry identifies and determines a molecule’s characteristics. Advances in spectrometry have allowed researchers to analyze very small protein samples. X-ray crystallography, for example, enables scientists to determine a protein crystal’s three-dimensional structure at atomic resolution. Another protein imaging technique, nuclear magnetic resonance (NMR), uses atoms’ magnetic properties to determine the protein’s three-dimensional structure in aqueous solution. Scientists have also used protein microarrays to study protein interactions. Large-scale adaptations of the basic two-hybrid screen have provided the basis for protein microarrays. Scientists use computer software to analyze the vast amount of data for proteomic analysis.

Genomic- and proteomic-scale analyses are part of systems biology, which is the study of whole biological systems (genomes and proteomes) based on interactions within the system. The European Bioinformatics Institute and the Human Proteome Organization (HUPO) are developing and establishing effective tools to sort through the enormous pile of systems biology data. Because proteins are the direct products of genes and reflect activity at the genomic level, it is natural to use proteomes to compare the protein profiles of different cells to identify proteins and genes involved in disease processes. Most pharmaceutical drug trials target proteins. Researchers use information that they obtain from proteomics to identify novel drugs and to understand their mechanisms of action.

Scientists are challenged when implementing proteomic analysis because it is difficult to detect small protein quantities. Although mass spectrometry is good for detecting small protein amounts, variations in protein expression in diseased states can be difficult to discern. Proteins are naturally unstable molecules, which makes proteomic analysis much more difficult than genomic analysis.

Source:

Clark, M., Douglas, M., Choi, J. Biology 2e. Houston, Texas: OpenStax. Access for free at: https://openstax.org/details/books/biology-2e


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