Molecular biology and subcellular fractionation

Molecular biology is a dynamic field dedicated to understanding the structure, function, and interactions of essential biological molecules, including DNA, RNA, and proteins. Central to this discipline is the investigation of how these molecules contribute to the organization and regulation of cellular components within various cellular compartments. Subcellular fractionation is a fundamental technique in molecular biology, enabling researchers to separate and analyze specific parts of the cell, such as mitochondria, nuclei, and cytoplasm. By isolating these compartments, scientists can study the unique protein composition of each, investigate the localization of mitochondrial proteins and membrane proteins, and gain insights into the intricate processes of cell biology. This approach is especially valuable in cultured cells, where understanding protein localization and function can reveal critical aspects of cellular regulation and disease mechanisms.

We offer a range of subcellular fractionation kits, which include all the required reagents and step-by-step protocols to isolate nuclear, cytoplasmic, mitochondrial, and membrane fractions with confidence and reproducibility.

Cell Fractionation Kit - Standard (ab109719) - Allows for the rapid and simple preparation of mitochondrial, cytoplasmic, and nuclear fractions from cultured cells of mammals and other species.

Mitochondria/Cytosol Fractionation Kit (ab65320) - Provides a unique formulation of reagents for the effective isolation of highly enriched mitochondrial fractions from the cytosolic fraction of mammalian cells, including both apoptotic and non-apoptotic cells.

Nuclear/Cytosol Fractionation Kit (ab289882) - Can be used for the isolation of nuclear/cytosol fractions without ultracentrifugation.

Cell Fractionation Kit - High Throughput (HT) (ab109718) - Provides a method and reagents for a rapid and straightforward preparation of cytosolic, mitochondrial, and nuclear fractions from adherent cells.

Fraction-PREP Cell Fractionation Kit (ab288085) - Designed to provide reproducible extraction of four subcellular protein fractions (Cytosol, nucleus, membrane/particulate, and cytoskeletal fractions) from a single mammalian sample.

Cell culture and preparation

The success of subcellular fractionation relies heavily on the careful preparation of cells, with cultured mammalian cell lines like HeLa cells being widely used for these studies. Proper cell culture techniques ensure that cells are healthy and suitable for downstream applications. The preparation process begins with harvesting the cells, followed by thorough washing to remove any residual media or contaminants. Cell lysis is then performed under controlled conditions to preserve the integrity of cellular components, particularly when isolating intact mitochondria and minimizing cross-contamination between subcellular fractions. The choice of lysis buffer and method is crucial, as it affects the yield and purity of the resulting fractions. Differential centrifugation is subsequently employed to separate the various cellular compartments based on their size and density, allowing for the isolation of soluble cytosolic proteins, membrane-bound proteins, and other cellular components. This meticulous approach ensures that each subcellular fraction accurately represents its respective compartment, providing reliable material for further molecular biology analyses.

Last edited Wed 14 Oct 2020

Materials

Subcellular fractionation buffer

​Just before use, add the following per 10 mL:

Stage 1 - Procedure for cultured cells

All centrifugations should be done at 4°C. Samples should be kept on ice throughout the procedure.

Steps

Transfer cells from 10 cm plates into 500 μL fractionation buffer, eg by scraping. Incubate for 15 min on ice.

Using a 1 mL syringe, pass the cell suspension through a 27-gauge needle 10 times (or until all cells are lysed).

Leave on ice for 20 min.

Centrifuge the sample at 720 x g for 5 min. The pellet will contain the nuclei, and the supernatant will contain the cytoplasm, membrane, and mitochondria.

Transfer the supernatant into a fresh tube and keep on ice. This will be used further in Steps 8–11.

Wash the nuclear pellet from Step 4 with 500 μL fractionation buffer.

• Disperse the pellet with a pipette and pass it through a 25-gauge needle 10 times. Centrifuge again at 720 x g for 10 min. Discard the supernatant and keep the pellet containing the nuclei.

Resuspend the pellet from Step 6 in TBS with 0.1% SDS.

• Sonicate the suspension briefly to shear genomic DNA and homogenize the lysate (3 sec on ice at a power setting of 2-continuous).

Centrifuge the supernatant recovered in Step 5 at 10,000 x g for 5 min.

• The pellet contains the mitochondria. Transfer the supernatant into a fresh tube and keep on ice: this is the cytoplasm and membrane fraction.

Process the mitochondrial pellet from Step 8, as described for the nuclear pellet in Step 7, to obtain mitochondrial lysate in TBS/0.1% SDS.

For the membrane fraction, centrifuge the supernatant from Step 8 in an ultracentrifuge at 100,000 x g for 1 h.

• Wash the pellet by adding 400 μL of fractionation buffer. Resuspend by pipetting and pass through a 25-gauge needle. Re-centrifuge for 45 min. Resuspend the membrane pellet in the same buffer as used for the nuclei.

Optional: concentrate the supernatant

• Concentrate the supernatant by centrifuging through the filter unit. This concentrates the cytosol down to approximately 50–75 μL.