The overall goal of this experimental procedure, is to optimally homogenize cultured cells and to analyze partitioning of cellular proteins between membrane and soluble fractions. This method can help address one of the key challenges in cell biology, that is the subcellular compartmentalization of membrane peripheral proteins. The main advantage of this technique is, it can reproducibly detect partitioning of endogenous proteins, inside a solid and membrane fractions, with great accuracy and without use of detergents.
To begin, culture HGK293 cells at 90%confluency, such that the yield per one 15 centimeter dish is about 2 x 10 to the 7th cells when cultured in DMEM at 37 degrees celsius, with 5%carbon dioxide. Then, aspirate the growth medium by vacuum. Next, place the culture dishes on ice and gently wash the cells twice on the culture dishes with 10 milliliters of chilled homogenization buffer per dish.
After washing, use a large cell scrapper to harvest the cells, using an appropriate volume of homogenization buffer. Now, transfer the cell suspension to a clean, pre-chilled self destruction bomb, placed in an ice bath on a stir plate. To maintain suspension homogeneity, place a micro magnetic stir bar inside the bomb and turn on the stir plate.
After that, add one protease inhibitor tablet to the suspension and close the bomb according to the manufacturer's instructions, including a crucial step of pushing the O ring upward to secure proper closing of the bomb. Once done, pressurize the bomb gradually with the nitrogen gas tank, as per the manufacturer's instructions, until the pressure gauge reads between 300 to 600 PSI. After the desired pressure is obtained, close all valves and disconnect the nitrogen tank.
Allow 20 minutes for the nitrogen to dissolve and reach equilibrium within the cells. Next, use a disposable wipe to remove excess water around the discharge valve, and gently open the valve to achieve a drop wise release of homogenate, collect the homogenate in a pre-chilled 15 milliliter tube. Observe the milky appearance with foam on top of the cavitate.
During the pressurization, it is critical to avoid simple loss by collecting the cavitate with slow, valvular discharge into a collection tube with an adequate access volume capacity that is optimally positioned to accommodate sudden discharge near the end of collection. After collecting the cavitate, centrifuge the tubes to remove unbroken cells in nuclei. And then collect the post-nuclear supernatant or PNS.
Next, transfer the PNS to a polycarbonate ultracentrifuge tube. Perform ultracentrifugation to separate the cytosolic and membrane fractions. After ultra centrifugation and separation of pellet and supernatant.
Use a one milliliter pipette to collect the supernatant or S fraction. After collecting the supernatant, rinse the pellet carefully with four milliliters of cold PBS, taking care not to disturb pellet and then remove the buffer by aspiration. Re-suspend the pellet, using the same volume of solubilization buffer, as the cytosolic fraction to achieve cell equivalence.
Next, centrifuge the fully solubilized pellet suspension in a tabletop centrifuge. Use a one milliliter pipette to collect the supernatant or P fraction and discard the pellet. Finally, store the fractions at 80 degree celsius.
Endogenous protein levels from both cytosolic and membrane fractions, isolated from three representative cell lines, are shown after western blot analysis. The presence of loosely associated peripheral membrane proteins such as, EEA1, Rab7, Rab9 and hexokinase 1 in both cytosolic and membrane fractions, demonstrate the utility of this technique and evaluating the strength of the membrane association of peripheral proteins. Via mounts of endogenous proteins in the PNS, were analyzed after subjecting jurkat cells to different mechanical disruption techniques.
The yield of peripheral membrane proteins such as, hexokinase 1 and Ras, is higher in samples prepared with nitrogen cavitation. The homogenisation efficiency was compared between a hypotonic buffer and a buffer made isotonic with sucrose or sodium chloride. The protein levels in the PNS of cells disrupted using isotonic sucrose buffer, showed less recovery except for fibrollarin, which was enriched.
These results indicate the partitioning of farnesylated NRAS into both P and S fractions. The absence of the preno modification of NRAS results in entirely cytosolic localisation, which therefore, confirms the reliability of this protocol for studying the dynamics of peripheral membrane protein partitioning. Once mastered, the separation of cytosolic and membrane fractions without re-suspension of the membrane fraction can be performed in an hour and 45 minutes or about 4 hours with re-suspension of the membrane fraction.
Due to the inherent artifactual nature of bichemical fractionation, it is important to focus on the changes across experimental conditions in the partitioning of proteins, rather than the absolute distribution between cytosolic and membrane fractions. Additionally, we suggest testing different homogenization buffers for nitrogen cavitation to achieve optimal results. Post this procedure, immunoblotting and enzyme activity assays can be performed in order to answer additional questions like, membrane associated strength of peripheral proteins.
Don't forget that working with high pressure nitrogen bombs can be extremely hazardous and precautions such as, wearing appropriate personal protection should always be taken while performing this procedure. Thanks for watching, good luck with your experiments.
