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Subcellular Fractionation Protocol

By Sean Mac Fhearraigh PhD

Isolation of nuclear, cytosolic and mitochondrial fractions of high quality purity from mammalian tissues allows for different cellular proteins and organelles to be studied and characterised. Subcellular fractionation can be used for a wide variety of cell types for sample preparation before omics analysis.The protocol described below has been designed and optimized for the subcellular fractionation of nuclear, mitochondrial and cytoplasmic extracts from cells using a sucrose gradient. Depending on the number of samples you have this protocol can take up to 3 – 4 hours to complete. You can use this protocol as a starting point for the subcellular fractionation of other cellular samples although you may need to change the buffer volumes, homogenization duration and intensity etc.

Components of a subcellular fractionation Protocol

1.Tissue Disruption and Lysis

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. 3 basic methods can be used for tissue and cell lysis 1) homogenization, 2) sonication, and 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.

2. Centrifugation

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.

3.Differential Centrifugation.

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.

Growth and maintenance of chronic myeloid leukaemia cells lines

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%).

Isolation of mitochondria – Procedure

  1. Culture K562 cells (1 x 107) in a T175 cm² tissue culture flask.
  2. Harvest cells by centrifugation at 270 x g for 5 min.
  3. Aspirate supernatant
  4. Resuspend cells in 1 ml of ice cold PBS
  5. Centrifuge at 270 x g.
  6. Resuspend the pellet in 300 µl of sucrose cell extraction buffer (SCEB) (see below recipe)
  7. Allow cells to equilibrate on ice for 30 min, prior to rupture by 25 strokes through a 25-G needle.
  8. Centrifuge cells at 2000 x g for 3min at 4 °C
  9. Transfer the supernatant containing intact mitochondria and cytosolic protein to a new microcentrifuge tube.
  10. Resuspend the unbroken pelleted cells 100 µl SCEB and disrupt further by passaging through a 30G needle X 10.
  11. Centrifuge cells 2000 x g for 3 min.
  12. Repeat the titration step
  13. Pool the cell fraction containing the mitochondria and cytosolic.
  14. Resuspend the remaining pellet of broken cells and debris in RIPA buffer and stored at -20 °C for further analysis.
  15. Centrifuge the pooled mitochondria and cytosolic protein at 2000 x g at 4 °C for 3 min to pellet unbroken cells and nuclei.
  16. Repeat twice each time adding the supernatant to a fresh eppendorf.
  17. Centrifuge the supernatant at 7,000 x g at 4 °C for 10 min to pellet whole mitochondria.
  18. Resuspend the mitochondrial pellet in SCEB buffer and left on ice for 10 min
  19. Transfer the supernatant containing the cytosolic protein to a fresh eppendorf.
  20. Centrifuge the mitochondrial fraction at 7,000 x g for 10 min and lyse in RIPA buffer.
  21. Determine the protein concentration using a Bradford assay
  22. Store samples at -20 °C.

Isolation of K562 cell nuclear fractions – Procedure

  1. Culture K562 cells (5 x 107) in a T175 cm² tissue culture flask.
  2. Lyse cells in 2.5ml of Homogenising buffer (HB) on ice for 15 min (see below Recipe)
  3. Aliquot cells (500 µl) to a fresh micro-centrifuge tube
  4. Rupture by X 25 strokes through a 25-G needle.
  5. Centrifuge cells at 600 x g for 10 min at 4 °C
  6. Remove the supernatant containing the mitochondrial and cytosolic protein fractions
  7. Resuspend the pellet containing the nuclear fraction in 250 µl of HB and centrifuge at 600 x g for 10 min.
  8. Remove and discard the supernatant.
  9. Resuspend the pellet in a 2.2 M sucrose solution containing 1 mM MgCl2 and 10 mM Tris-HCl, pH 7.4 and layered over 1 ml of 2.2 M sucrose solution.
  10. Centrifuge at 80,000 x g for 80 min using a SV-T55i rotor.
  11. Following centrifugation, decant the supernantant and clean the sides of the centfifuge using an ethanol soaked tissue.
  12. Resuspend the nuclear pellet in 200 µl of HB
  13. Centrifuged at 600 x g for 10 min x2 times to remove any possible mitochondrial or cytosolic contamination.
  14. Resuspend the nuclear pellet in RIPA buffer.
  15. Store samples at -20 °C.

Sucrose Cell Extraction Buffer (SCEB) Recipe

  1. 300 mM sucrose,
  2. 10 mM Hepes, pH 7.4,
  3. 50 mM KCl,
  4. 5 mM EGTA,
  5. 5 mM MgCl2

Proteinase and Phophatase inhibitors

  1. 1 mg/ ml aprotinin,leupeptin, pepstatin
  2. 25.5 mM NaF,
  3. 1 mM Na3Vo4,
  4. 20.5 mM beta-glycerophosphate
  5. 100 µM PMSF

Homogenising buffer (HB) Recipe

  1. 0.25 M Sucrose,
  2. 10mM Tris-HCL, pH 7.4,
  3. 5mM MgCl2

Proteinase and Phophatase inhibitors

  1. 1 mg/ml aprotinin, leupeptin, pepstatin,
  2. 25.5mM NaF,
  3. 1 mM Na3VO4,
  4. 20.5 mM beta-glycerophosphate
  5. 100 µM PMSF

Purity and Validation of subcellular fractionation

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.