Frustrated by the time and energy consuming process of subcellular fractionation, I had wondered if there was an easier method available. I found out about the Chemicon Compartmental Protein Extraction Kit and after trying it out, found that it worked well for my applications. In my research, I perform subcellular fractionation (particularly, cytoskeleton fractionation) from hippocampal slices and detect related changes in target protein expression and localization after drug treatment. With this kit, I obtained four fractions containing cytoplasmic, nuclear, membrane and cytoskeletal materials. I tested the efficiency of the compartmental protein purification by Western Blot, using different antibodies for subcellular marker proteins. I used GAPDH for the cytoplasmic, Arc for cytoskeleton, Histone H4 for nuclear and GluR1 for membrane fraction markers. Results were successful and each marker was saturated in its respective fraction.
The kit contains five buffers: buffer C for homogenization and cytoplasmic protein extraction, buffer W for washing after the first cytoplasmic extraction, buffer N for nuclear protein extraction, buffer M for membrane protein extraction and buffer CS for cytoskeletal protein extraction. They also provide 0.55 ml of 50x protease inhibitor cocktail. Before the experiment starts, the 50x protease inhibitor cocktail is added to the buffers. The experimental procedure is very simple and straightforward. It consists of five protein extraction steps and supernatant collection. After the collection of the cytoskeletal protein fraction, you will get a viscous pellet of genomic DNA (should not affect the compartment protein extraction). The whole procedure will take around 3 hours.
This compartmental protein extraction kit provided a novel, easy to perform and inexpensive method to sequentially isolate cytoplasmic, nuclear, membrane, and cytoskeleton proteins. Detection applications include differential subcellular localization of targets, subcellular targeting shift and post-translational modification of low abundance proteins. However, caution needs to be used for certain proteins. For example, some cytoskeleton proteins may not be detected in the cytoskeleton protein fraction, depending on how strong the binding is to the cytoskeleton. Also, you may get cross-contamination between subcellular fractionations, such as nuclear proteins in the cytoplamic fraction, so you need to optimize the homogenization condition for your tissue or cells. It is worthy to mention that the kit comes with a very detailed protocol and trouble-shooting information, which is quite helpful.
Fen Huang
Graduate Student
Department of Anatomy and Neurobiology
University of California, Irvine