Encyclopedia of Experiments: Cancer Research
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Transcript
- In leukemic cells, the shuttling of macromolecules, such as a few tumor suppressing proteins between the nucleus and the cytoplasm, is deregulated. Mislocalization of these proteins makes them inactive, resulting in the uncontrolled growth of leukemic cells. We can determine the aberrant location of such proteins by generating the cytoplasmic and nuclear fraction from leukemic cells.
Start by taking the desired volume of leukemic cells in a microfuge tube and centrifuge to pellet the cell. Re-suspend the pellet in a hypotonic lysis buffer and then add the desired concentration of detergent. Hypotonic lysis buffer causes the cells to swell up and break the plasma membrane, leaving the nuclear membrane intact, thus isolating cytoplasmic fractions. The detergent helps in the extraction of proteins from cytoplasmic organelles.
Centrifuge the lysed cells in suspension and transfer the supernatant to a fresh tube. The supernatant contains the cytoplasmic fraction of the cell. To prepare the nuclear fraction, resuspend the remaining pellet in the complete lysis buffer and add detergent. Centrifuge and collect the nuclear fraction present in the supernatant into a microfuge tube. Store the cytoplasmic and nuclear fractions at minus 80 degrees Celsius for further analysis. In the following protocol, we will demonstrate the generation of subcellular fractions from leukemic cells.
After stimulation and/or drug treatment incubation is complete, transfer of the cells into individually labeled 1.5 milliliter microfuge tubes and pellet by centrifuging at 200 times g for 5 minutes at 4 degrees Celsius. After discarding the supernatant, resuspend the cell pellet in 1 milliliter of ice cold PBS with phosphatase inhibitors. Centrifuge at 200 times g for 5 minutes at 4 degrees Celsius. Remove the supernatant and keep these whole cell extract pellets on ice for further preparations.
To prepare cytoplasmic fractions, gently resuspend the cell pellets in 50 microliters of 1 X hypotonic buffer. To allow the cells to swell, incubate them on ice for 15 minutes. After performing a detergent gradient to determine the optimal concentration of detergent to use for a specific cell type, add 0.8 to 2.5 microliters of detergent into each sample and vortex on the highest setting for 10 seconds.
To verify cell lysis, observe the cells under a phase contrast microscope before and after addition of detergent. All cells have a dense dark nucleus, surrounded by the cytoplasm that appears as a bright halo. Once lysed, centrifuge the samples at 14,000 times g for 30 seconds at 4 degrees Celsius. Carefully, without disturbing the pellet, transfer the supernatant into a pre-chilled, labeled microfuge tube and store this cytoplasmic fraction at minus 80 degrees Celsius until further analysis.
Ensure the complete removal of the cytoplasmic fraction to prevent contamination of the nuclear fraction.
To the remaining pellet, which contains the nuclear fraction, add 50 microliters of complete lysis buffer and resuspend by pipetting up and down. To solubilize proteins associated with the nuclear membrane, add 2.5 microliters of detergent, vortex on the highest setting for 10 seconds, and incubate on ice for 30 minutes. Vortex on the highest setting for 30 seconds and centrifuge. Then, transfer the supernatant into a pre-chilled, labeled microfuge tube, and store this nuclear fraction at minus 80 degrees Celsius until further analysis.
