Protein Purification and Crystallization

Liquid column chromatography (LC) is our standard method of choice to purify proteins. Using the characteristics of our different proteins like size or charge gives us the changes to separate them from each other. The principle of LC relies on different strong interactions between the proteins and the column material resulting in different long retention times.     Our LCC equipment consists of:

• one Äkta pure

• one Äkta purifier

• one Amersham FPLC

• three Äkta explorer

Another reliable purification method is density gradient centrifugation. Using a gradient of densities caused by different concentration of e.g. sucrose or glycerol a separation by the density of your sample is achieved. After centrifuging the gradient with sample on it in ultracentrifuge we hopefully obtain bands of the protein of interest and other impurities. 

Working and purifying proteins can sometimes cause relatively harsh conditions for the proteins. To check whether the purified proteins is still capable we are using a Clark electrode. Measuring the oxygen evolution for PSII and oxygen consumption for PSI after light excitation gives us a clear idea how our protein is doing. Only active protein can be still used for further experiments. Our lab uses the Hansatech Oxylab+.

To verify the success of purification gelelectrophoresis is an often used method to check for impurities,  oligomeric states and/or subunits. We are using Blue Native PAGE to check our sample in native manner. The idea is to see whether we still have other oligomeric states inside the sample. With SDS PAGE our sample get denaturated resulting in a pattern of subunits only. Our lab uses the gel equipment Mini-PROTEAN® from Bio-Rad. 

The crystallization of purified protein is mostly our last sample preparation step before the actual experiment. A buffer condition has to be found where protein leaves the solution and forms uniform sized and shaped crystals. The resulting lattice of highly arranged order protein molecules next to each other makes it possible to reliably measure the actual structure of your protein of interest.