Subcellular Fractionation Protocol - Mitochrondria, Nuclear, Cytosolic

The first step in cell fractionation is tissue disruption and cell lysis. This step allows you to dis-aggregate and break open the cells with minimal damage to the cellular fraction of interest. Three basic methods can be used for tissue and cell lysis:

1. Homogenization,
2. Sonication,
3. Osmotic lysis.

The method you use depends on cell type and the subcellular fraction of interest. The most commonly used method for animal and plant tissues is homogenization. This process involves the use of a mechanical homogenizer acting like a pestle and mortor/blender to break the tissue apart and lyse cells. To disrupt and lyse prokaryotic cells sonication is used. This process uses ultrasound to disrupt cellular membranes.

Osmotic lysis is used when the cells of interest are vulnerable to osmotic stress, such as red blood cells.

Following the initial disruption and lysis of cells, centrifugation is carried out. Centrifugation ensures the cellular components settle at the bottom of the tube. During centrifugation, the lysate is rotated at a certain speed, known as RPM – rotations per minute. This rotation imposes a force on the particles perpendicular to the axis of rotation and is known as RCF – relative centrifugal force, expressed as a multiple of the force of Earth’s gravitational force (x g). The part of the centrifuge that holds the centrifugation tubes is called the centrifuge rotor. Centrifuges can have a number of different rotors. There are three types of centrifuge rotors: fixed angle rotors, swinging bucket rotors, and vertical rotors.

Fixed-angle and swinging-bucket rotors are the most commonly used. As the name suggests in a fixed-angle rotor the centrifuge tubes are spun at a fixed angle, which is ideal for pelleting cells and subcellular components. Swinging-bucket rotors enables the tubes to swingout perpendicular to the axis of rotation as the rotor rotates and is commonly used in density-gradient centrifugation schemes.

Differential centrifugation is the sequential centrifugation of a cell lysate at progressively increasing centrifugation force, isolating cellular components of decreasing size and density. The separation of the cellular components is based on their sedimentation rate through the centrifugation medium and therefore is dependent on the size and shape of the cellular components.

Differential centrifugation results in the production of a pellet following each centrifugation step. The pellet contains a mixture of cellular components of roughly the same size and density. Following each centrifugation step you can remove the supernatant and centrifuge again, allowing you to pellet other cellular components of a lesser size and density.

Cell lines used in the study include the K562 and LAMA84 chronic myeloid leukaemia cells and HeLa cervical cancer cells. All cell lines were provided by the European Collection of Cell Cultures (ECACC). K562 and LAMA84 cells were grown in suspension in RPMI 1640 medium supplemented with 2 mM L-glutamine, penicillin (100 µg/ ml), streptomycin (100 µg/ ml) and foetal bovine serum (FBS) (10% (v/v)). HeLa cells were grown in Dulbecco’s modified eagles media supplemented with 2 mM L-glutamine, penicillin (100 g/ ml), streptomycin (100 µg/ ml) and foetal bovine serum (FBS) (10% (v/v)). Cells were incubated at 37°C, with CO2 (5%) and O2 (95%).

The purity of your isolated fractions can be determined using immunoblotting for specific protein markers such as histone H3 (nuclei) and cytochrome oxidase IV (CoxIV, mitochondria). Validation of the purity of the subcellular fractions derived from the same starting sample can be determined by examing “house-keeping” (HK) protein markers via western blot analysis.

Separate the protein samples using SDS-PAGE and transfer to a nitrocellulose membrane. This membrane can then be probed using selected monoclonal antibodies such as Histone H3, GAPDH or Cox IV for nuclei, cytosolic and mitochondrial HK fractions respectively. To visualize your proteins a secondary a specific secondary HRP-conjugated antibody is added and the blot exposed.