A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue
© Dimauro et al.; licensee BioMed Central Ltd. 2012
Received: 5 April 2012
Accepted: 31 August 2012
Published: 20 September 2012
We describe a method for subcellular fractionation of mouse skeletal muscle, myoblast and myotubes to obtain relatively pure fractions of nuclear, cytosolic and mitochondrial compartments. Fractionation allows the analysis of a protein of interest (or other cellular component) based on its subcellular compartmental distribution and can also generate molecular information about the state of a cell and/or tissue and how the distribution of a protein may differ between different cellular compartments, tissues or cell types, in response to treatments or ageing.
The described method was specifically developed for skeletal muscle and proliferating/differentiated muscle cells. The purity of the different fractions, representing the cytoplasmic, mitochondrial and nuclear subcellular compartments was validated by western blot analysis of “house-keeper” marker proteins specific for each cellular compartment.
This low cost method allowed the mitochondrial, cytoplasmic and nuclear subcellular compartments from the same starting muscle samples to be rapidly and simultaneously isolated with good purity and without the use of an ultracentrifuge. This method permits samples to be frozen at −80°C for future analysis and/or additional processing at a later date.
Isolation of nuclear, cytosolic and mitochondrial fractions of reasonable purity from mammalian tissues and cells has generated great interest as it has the advantage of allowing different cellular proteins and organelles to be studied and characterised. Subcellular fractionation is universally used for various cell types and tissues for sample preparation and prior to subsequent ~ omics analysis [1–5]. Generic fractionation protocols exist that can purify specific subcellular compartments and organelles, but in general they are not tailored for use with skeletal muscle and may require large amounts of starting material, time, or special reagents whilst potentially yielding fewer fractions from the same starting sample etc. [3, 5–9]. The protocol described has been optimized for use with primary skeletal muscle tissue (e.g. mouse anterior tibialis (AT) muscle) and both proliferating and differentiated C2C12 cells to isolate subcellular fractions of nuclei, cytosol, and mitochondria from a single starting sample, thereby reducing the quantity of starting material, cost and total time needed for sample preparation.
The protocol works well for skeletal muscle tissue and cells and could be used as a starting point for the fractionation of other non-muscle samples although changes to buffer volumes; homogenization duration/intensity etc. may be required. The purity of the fractions obtained was assessed by immunoblotting for specific protein markers: histone H3 (nuclei), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, cytosol), and cytochrome oxidase IV (CoxIV, mitochondria).
Cell culture and animals
The C2C12 mouse skeletal myoblast cell line was obtained from the American Type Culture Collection (CRL-1772). C2C12 myoblasts were maintained in DMEM (Sigma Aldrich, Poole, UK) supplemented with 1% L-glutamine (Lonza, Cologne, Germany), 10% FBS (Biosera, Sussex, UK) and 1% penicillin and streptomycin (Sigma) under an atmosphere of 5% CO2 in humidified air at 37°C. To induce myogenic differentiation, the growth medium was changed to differentiation medium (DMEM supplemented with 2% horse serum (Sigma) and 1% antibiotics) after myoblasts had reached ≈ 90% confluence in a T75 cm2 flask. Myoblast cells were either harvested at 90% confluence or allowed to mature to myotubes for 7 days and then harvested (see below).
Adult mice (C57BL/6) were euthanized by overdose with anesthetic (ketamine hydrochloride and medatomidine hydrochloride) administered by intraperitoneal injection. Anterior tibialis (AT) muscles, approximately 50 mg wet weight, were rapidly removed and used fresh to prepare fractions. Experiments were performed in accordance with UK Home Office Guidelines under the UK Animals (Scientific Procedures) Act 1986 and received ethical approval from the University of Liverpool Animal Welfare Committee.
The pellet P0 (containing nuclei and debris) was resuspended in 300-500 μl STM buffer, vortexed at maximum speed for 15 seconds and then centrifuged at 500 g for 15 minutes. Following the above step, the nuclear pellet was labelled as P1 and kept on ice, the supernatant S1 (cell debris) was discarded. The purity of the nuclei within fraction P1 can be quickly determined by microscopic inspection by diluting an aliquot of the fraction in a trypan blue solution on a haemocytometer. If the P1 fraction contained excess cell debris the above step was repeated once.
To increase the P1 fraction purity further it was washed in STM buffer (300-500 μl), vortexed at maximum speed for 15 seconds and then centrifuged at 1,000 g for 15 minutes. The washed pellet was labelled as P5 (S5 was discarded) and resuspended in 200-500 μl NET buffer (comprising: 20 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.5 M NaCl, 0.2 mM EDTA, 20% glycerol, 1% Triton-X-100, protease and phosphatase inhibitors) using a pipette to triturate until homogeneous. Pellet P5 was vortexed at maximum speed for 15 seconds and incubated on ice for 30 minutes, this fraction contained the nuclei. The nuclei were lysed with 10–20 passages through an 18-gauge needle and/or sonicated (using a Soniprep 150, MSE, London, UK) at high setting for 10–15 seconds with 30 second pauses whilst being kept on ice throughout. The lysate was centrifuged at 9,000 g for 30 minutes (at 4°C), the resultant supernatant (S6) was the final “nuclear fraction” (Figure 1).
Cytosolic and mitochondrial fractions were extracted from S0 by centrifugation at 800 g for 10 minutes. The supernatant S2 was saved and the pellet (P2) was discarded, though to improve the nuclear yield the pellet P2 can be combined with fraction P0 (optional step). S2 was then centrifuged at 11,000 g for 10 minutes and the supernatant S3 (containing cytosol and microsomal fraction) was precipitated in cold 100% acetone at −20°C for at least 1 hour followed by centrifugation at 12,000 g for 5 minutes, the pellet (P7) was then resuspended in 100-300 μl STM buffer and labelled as “cytosolic fraction” (Figure 1) that likely included some microsomal content. The pellet P3 was again resuspend in 100-200 μl STM buffer and centrifuged at 11,000 g for 10 minutes. Once centrifuged, supernatant S4 was discarded, the mitochondrial pellet (P4) was resuspended in 50-100 μl SOL buffer (comprising: 50 mM Tris HCl pH 6.8, 1 mM EDTA, 0.5% Triton-X-100, protease and phosphatase inhibitors) by sonication on ice at high setting for 5–10 seconds with 30 second pauses and labelled as “mitochondrial fraction”. All buffers and centrifugation steps were modified from Cox and Emili  and Psarra et al. .
Additionally, a commercial cell fractionation kit designed to yield near pure nuclei and cytoplasmic fractions from the same starting sample of cells and soft tissues was used (Thermo NE-PER nuclear and cytoplasmic extraction kit, Pierce-Thermo, Northumberland, UK, to obtain a mitochondrial fraction required the use of an additional kit) as directed by the manufacturer. This enabled a comparison of the efficacy of the commercial kit with the method described here when both methods were used to fractionate an identical starting sample (myoblast cells from a T75 cm2 flask, approximately 2×106 cells).
The protein content of each compartment was determined using BCA protein assay (Sigma).
Fractionation validation using western blotting
Results and discussion
Fraction yields for myoblast, myotube and AT tissue
Mean protein yield (μg)
Total sample protein (mg)
No. of replicates
1179 ± 100 (150μL)
496 ± 25 (100μL)
97 ± 13 (50μL)
1.8 ± 0.1
1624 ± 107 (150μL)
547 ± 4 (100μL)
197 ± 15 (50μL)
2.4 ± 0.2
3294 ± 254 (300μL)
1486 ± 225(300μL)
444 ± 60 (100μL)
5.2 ± 0.5
Purity analysis of the yield of sub-compartmental marker proteins
Nuclei/Histone H3 (%)
92.7 ± 1.3
86.4 ± 1.4
88.0 ± 0.9
92.4 ± 0.5
93.9 ± 1.4
87.8 ± 0.3
85.6 ± 0.9
92.1 ± 0.5
91.2 ± 0.1
In comparison with the described protocol, the yield obtained with the commercial kit as indicated by band density, was poor and only the cytoplasmic fraction appeared relatively pure. The kit is designed for more generic use with multiple cells and soft tissues and may explain the inability to generate a pure and abundant nuclear fraction from muscle. The main advantage of the kit was a slightly reduced processing time although an additional kit to purify a mitochondrial (and cytoplasmic) fraction would be required and this would increase the cost and time requirement.
In conclusion the method for subcellular fractionation described here is inexpensive, does not require an ultracentrifuge and was found to generate three relatively abundant subcellular fractions of reasonable purity. This method of subcellular fractionation could be combined with proteomics research wherein protein patterns of subcellular fractions could be mapped and characterized by 2D gel analysis and mass spectrometry.
Ivan Dimauro and Timothy Pearson joint first author.
Funding by the MRC (TP, MJJ) and University of Rome “Foro Italico” (ID, DC) are gratefully acknowledged.
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