Purification and analysis of colorful hypothetical open reading frames: An inexpensive gateway laboratory

This laboratory exercise is an inquiry-based investigation developed around the core experiment where students, working alone or in groups, each purify and analyze their own prescreened colored proteins using immobilized metal affinity chromatography (IMAC). Here, we present reagents and protocols that allow 12 different proteins to be purified in parallel without specialized equipment and within a 2.5- to 3-hour undergraduate teaching laboratory. The visual feedback of purifying a colored biomolecule provides real-time emphasis of the power and simplicity of recombinant DNA technology and IMAC. As presented here in its simplest form, this laboratory occupies two laboratory periods: purification followed by SDS-PAGE analysis. As such, it can be easily inserted into the existing curriculum of a Biochemistry, Molecular Biology, Biotechnology, or even Genetics course to illustrate core concepts of central dogma and protein purification. Furthermore, the proteins in hand at the end of this 2-week module can also be used for follow-up experiments tailored to the needs, timeframe, and facilities available.