Whole cell protein analysis is an excellent way to assess protein content of a specific cell population. Nevertheless, the shift of proteins into different compartments due to activation, differentiation or other mechanisms needs to be analyzed in a more sophisticated way. Fractionation of subcellular proteins enables protein localization assessment and protein enrichment from specific cellular compartments.
The Subcellular Protein Fractionation Kit from Pierce contains sufficient reagents for extracting 50 cell pellet fractions having a packed cell volume of 20 ul each. The kit comes with all the necessary extraction buffers for the cytoplasmic, membrane, nuclear, and cytoskeletal fractions. In addition, using the enclosed micrococcal nuclease, chromatin-bound nuclear proteins can get released and isolated. A protease inhibitor cocktail to protect from unwanted protein degradation is included, too. Additional materials required are PBS, a cooled microcentrifuge and a rotary shaker in the cold room or in the fridge.
I used the kit for subcellular fractionation of platelets as well a megakaryocytes. The samples were used for different downstream applications, especially 2D electrophoresis or Western blot analysis. The included protocol is very easy to follow, straightforward, fast, and a very informative troubleshooting table is included. The kit is also compatible with a variety of downstream applications, like Western blot, different protein concentration assays, EMSA reactions, enzyme activity assays and 2D electrophoresis.
The protocol is a step-by-step approach using different lysis buffers. One thing to be careful about is the use of extraction buffers at different temperatures. (This could be highlighted in a more obvious way in the protocol.) After harvesting the cells, the first extraction buffer for the cytoplasmic proteins is added to the cell pellet. Following an incubation period and subsequent centrifugation, the supernatant containing the cytoplasmic proteins can be frozen. The use of the membrane extraction buffer will result in a fraction of membrane associated proteins. The next step, using the nuclear extraction buffer, will result in the isolated soluble nuclear protein fraction. By adding micrococcal nuclease to the same extraction buffer, the chromatin-bound protein fraction can be isolated after a simple incubation (15 minutes at 37ºC) and centrifugation step. Since the last centrifugation step was performed at the highest setting of the microcentrifuge, the remaining firm pellet will undergo a harsh lysis. The pellet extraction buffer will solubilize this pellet during an incubation period at room temperature. After another centrifugation step, the resulting supernatant containing the cytoskeletal extract can be transferred to a new tube. Therefore, the use of the kit will result in five distinct cellular fractions. Using a 2D approach there was some overlap between the fractions which is not surprising since a lot of proteins are localized in different compartments throughout the cell. Testing for more compartment specific proteins shows a good separation of subcellular fractions.
I think the kit is working very well, is easy to use and shows a good separation of subcellular compartments.
Subcellular Protein Fractionation Kit From Thermo Scientific Pierce Protein Research Products
Easy to use, fast protocol, compatible with a variety of downstream applications, good fractionation.
Be careful with different extraction buffer temperatures which are required for a good result.
The Bottom Line
Useful product for subcellular localization of proteins and monitoring of shifts in protein content in different cellular compartments.