This article describes the process of purifying human norovirus P domain proteins using size exclusion chromatography. The method involves loading a bacterial extract onto a chromatography column and monitoring the elution of proteins based on their size.
Take a size exclusion chromatography column packed with porous spherical gel beads.
Equilibrate the column with a buffer that preserves the structural integrity of the proteins during separation.
Load a purified bacterial extract containing recombinantly expressed human norovirus P domain proteins and residual protein impurities.
Add the buffer to drive the sample through the column.
Large residual impurities cannot enter the pores and pass through the spaces between the beads, eluting first.
Monitor elution using UV absorbance. A small peak indicates impurities exiting the column.
In contrast, the small P domain proteins enter the bead pores, travel a longer path, and elute later. A rise in the absorbance peak indicates the elution of the P domain proteins.
Collect the eluted fractions containing the P domain proteins, which are now ready for further analysis.
Wash the pumps and pipes of the high performance liquid chromatography purification system, and pre-equilibrate the size exclusion chromatography column with gel filtration buffer. Inject the P domain onto the column at a flow rate of one milliliter per minute, using a superloop or loop, depending on the volume of the concentrated sample. After the injection is finished, increase the flow rate to 2.5 milliliters per minute.
As the optical density increases, and the P domain comes off the column, collect fractions of 1.5 milliliters. The P domain is usually eluted as a dimer.
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