Proteins located within the cytoplasm play an important role in signal transduction pathways and glycolysis. They also act as intracellular receptors and form part of the ribosome, enabling protein synthesis. For many studies, the purification of these proteins is very important in order to gain important information about the behavior of cells. We have used the ProteoPrep® Universal Extraction Kit and obtained very promising results for a cytoplasmic protein for which we previously obtained lower yields when using other extraction methods.
The ProteoPrep® Universal Extraction Kit was designed through a collaboration of Proteome Systems and Sigma research scientists. The kit is intended to separate membrane and soluble cytoplasmic proteins. As this is a “universal” kit, it can be used with mammalian, bacterial or yeast cells; we used this kit with the bacterial or yeast cells. If using samples other than E.coli, the researcher should know how to disrupt the cells that he/she is wants to use, as some types of cells may required much more vigorous disruption.
The kit comes with 5 reagents: (a) soluble cyoplasmic extraction reagent, (b) soluble protein re-suspension reagent, (c) protein extraction reagent type 4, (d) tributylphosphine stock solution and (e) alkylating reagent, idoaceramide. The scale of the extraction can be adjusted to fit the number of cells or amount of tissue required. The reagents should be prepared fresh just prior to use.
The membrane extraction kit utilizes sodium carbonate which removes nearly all of the membrane-associated proteins, thus creating a membrane protein fraction. In samples more complex than bacteria, such as yeast, insect or mammalian cells and tissue, the nuclear and organelle proteins will be divided between the membrane and soluble fractions.
The only negative point that one can see with using this kit is that there are more steps in the procedure than conventional methods. The best feature of this kit is that you will get very good yields. Sigma claims that the special reducing and alkylating reagents used in the ProteoPrep® Universal Extraction Kit produce samples that exhibit improved focusing and decreased streaking in 2D gels. Running of 2D gels requires a highly purified protein; we believe this kit helps a lot to achieve that goal.
This ProteoPrep® Universal Extraction Kit generates two fractions of protein which are enriched for soluble proteins and membrane proteins and ready for application to immobilized pH gradient strips for two-dimensional electrophoresis. In summary, I feel this kit is one of best choices for isolatation of cyptoplasmic and plasma membrane proteins.