Preparation of Subcellular Fractions from Leukemic Cells: A Method to Generate Cellular and Nuclear Fractions from Leukemic Cells

- 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 t