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Purification of Mitochondria by Sucrose Step Density Gradient Centrifugation David A. Clayton and Gerald S. Shadel Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080028 Email Alerting Service Subject Categories
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Protocol
Purification of Mitochondria by Sucrose Step Density Gradient Centrifugation David A. Clayton1 and Gerald S. Shadel2,3,4 1
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147-2408; 2Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520-8023; 3Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520-8023
Mitochondrial fractions isolated from tissue culture cells or tissue such as liver after differential centrifugation can be purified further by density gradient centrifugation. Here we describe the use of sucrose for this purpose because it is commonly used and inexpensive and the resulting mitochondria preparations are useful for many purposes.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Mitochondrial suspension See Protocol: Isolation of Mitochondria from Tissue Culture Cells or Protocol: Isolation of Mitochondria from Animal Tissue (Clayton and Shadel 2014a,b).
MS homogenization buffer (1×) Sucrose step density gradient solutions Tris-HCl (5 mM)/EDTA (1 mM) Equipment
Centrifuge rotor (Beckman SW28 or equivalent) Centrifuge tubes (Ultra-Clear) Pasteur pipettes METHOD
1. Prepare 30-mL gradients by carefully layering 15 mL of 1.0 M sucrose step density gradient solution over 15 mL of 1.5 M sucrose step density gradient solution in Ultra-Clear centrifuge tubes. 4
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D.A. Clayton and G.S. Shadel
2. Carefully apply the mitochondrial suspension (7 mL total volume) on top of the sucrose layers. Do not overload the gradient. In general, the mitochondrial pellet from one mouse liver or 2 mL of tissue culture cells can be loaded onto one step gradient.
3. Centrifuge in an SW28 rotor, or equivalent, at 60,000g (22,000 rpm) for 20 min. The mitochondria will form a layer at the 1.0 M/1.5 M sucrose interface.
4. Remove the sample layer and its interface with the 1.0 M layer. 5. Collect the mitochondria with a Pasteur pipette using a gentle sweeping motion across the top of the 1.5 M layer. 6. Dilute the sucrose slowly and carefully to minimize the risk of osmotic rupture of the mitochondria. i. Add 5 mM Tris-HCl/1 mM EDTA drop-by-drop with gentle swirling. ii. Gradually increase the rate of dilution until a concentration of 0.25 M sucrose is reached. 7. Centrifuge the mitochondria at 7,000g–17,000g for 15 min. 8. Wash once with 30 mL of 1× MS homogenization buffer and resuspend in a buffer and volume appropriate for subsequent work. See Troubleshooting.
TROUBLESHOOTING Problem (Step 8): Mitochondria are damaged and/or the specific activity you are interested in does not
survive the procedure. Solution: There are many alternative methods for density gradient purification that may be better
suited for the use you intend. These include substituting Ficoll, Percoll, or iodinated media (OptiPrep, Nycodenz, and metrizamide) for the sucrose.
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Ficoll was one of the first media developed specifically for cell fractionation (de Duve 1971; de Duve and Beaufay 1981). It is most often used as a step gradient and is commonly used in the isolation of mitochondria and synaptosomes from brain. Percoll (polyvinylpyrrolidone-coated silica particles) can be used as a preformed discontinuous or continuous gradient, or as a self-generating gradient, in which the mitochondria can be banded isopycnically, with contaminating organelles buoyant at a slightly lighter density (higher in the tube). It is widely used for the separation of a variety of organelles. It is isoosmotic throughout the gradient and forms a self-generated gradient quickly. One drawback is that because it is a colloidal suspension, its removal may be problematic. Nycodenz and metrizamide are used as preformed continuous or discontinuous gradients. Nycodenz can form a self-generated gradient and has been used in this way to purify peroxisomes, but it does so very slowly. OptiPrep (Iodixanol) is a dimer of Nycodenz and has the advantage of forming self-generating gradients in a short period of time (3 h). Like Percoll, it is iso-osmotic throughout the gradient. The purification of mitochondria in OptiPrep (Graham et al. 1994) is interesting because at 100,000g a shallow gradient is formed in which peroxisomes form a pellet at the bottom, lysosomes and other contaminating organelles are buoyant in the steep portion of the gradient at the top, and the mitochondria are spread throughout the shallow portion of the gradient. Because no thick bands of organelles are formed, trapping is not a problem. After the top of the gradient is removed, the rest of the gradient containing the mitochondria is collected and pelleted. If necessary, the Optiprep can be removed by dilution and washing the mitochondria or by dialysis. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080028
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Purifying Mitochondria by Density Gradient Centrifugation
RECIPES MS Homogenization Buffer (1×)
210 mM mannitol 70 mM sucrose 5 mM Tris-HCl (pH 7.5) 1 mM EDTA (pH 7.5) The buffer should be ice cold before use. Sucrose Step Density Gradient Solutions
1.0 M sucrose or 1.5 M sucrose 10 mM Tris-HCl (pH 7.5) 1 mM EDTA (pH 7.5)
REFERENCES Clayton DA, Shadel GS. 2014a. Isolation of mitochondria from tissue culture cells. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080002. Clayton DA, Shadel GS. 2014b. Isolation of mitochondria from animal tissue. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080010. de Duve C. 1971. Tissue fractionation—Past and present. J Cell Biol 50: 20d–55d.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080028
de Duve C, Beaufay H. 1981. A short history of tissue fractionation. J Cell Biol 91: 293S–299S. Graham J, Ford T, Rickwood D. 1994. The preparation of subcellular organelles from mouse liver in self-generated gradients of iodixanol. Anal Biochem 220: 367–373.
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