仅供参考：Mitochondrial Protein Extraction:a) Collect cells by centrifugation at approximately 370g for 10 minutes. Decant supernatant and re-suspend cells in 10 packed cell volumes of NKM buffer (1 mM Tris-HCl pH 7.4; 0.13 mM NaCl; 5 mM KCl; 7.5 mM MgCl2).b) Pellet cells and decant supernatant, repeat this washing step 2 times. Resuspend cells in 6 packed cell volumes of homogenization buffer (10 mM Tris-HCl pH 6.7; 10 mM KCl; 0.15 mM MgCl2; 1 mM PMSF; 1 mM DTT; add PMSF and DTT immediately before use).c) Transfer cells to a glass homogenizer and incubate for 10 minutes on ice. Using a tight pestle, homogenize the cells. Check under microscope for cell breakage ( the optimum is around 60%).d) Pour homogenate into a conical centrifuge tube containing 1 packed cell volume of 2 M sucrose solution and mix gently. Pellet unbroken cells, nuclei and large debris at 1200 g for 5 minutes and transfer the supernatant to other tube. This treatment is repeated twice, transferring the supernatant to a new tube each time, discarding the pellet.e) Pellet the mitochondria by centrifugation at 7000 g for 10 minutes. Resuspend the mitochondria pellet in 3 packed cell volumes of suspension buffer (10 MM Tris-HCl pH 6.7; 0.15 mM MgCl2; 0.25 mM sucrose; 1 mM PMSF; 1 mM DTT). Spin at 9500 g for 5 minutes to re-pellet mitochondria.f) At this point you can add 1x protein loading buffer and run on a gel if a whole mitochondrial protein extract is needed, further purify the mitochondria on a sucrose gradient if a very pure extract is needed. To purify the soluble (S-100) mitochondrial fraction see protocol below.Soluble (S-100) Mitochondrial Protein Extraction:a) Ressupend mitochondria in 1/3 the packed cell volume lysis buffer (25 mM HEPES-KOH pH 7.6; 5 mM MgCl2; 0.5 mM EDTA; 10% Glycerol; 1 mM DTT; 1 mM PMSF). Put the suspension into a glass homogenizer and homogenize with a tight pestle. Add 0.5% Tween 20 and 0.5 M KCl.b) Incubate the mixture on ice for 5 minutes. The homogenization is repeated 10 times.c) Spin the final mitochondrial lysate at 100,000 g in a ultracentrifuge at 4?C for 60 minutes.d) Carefully collect the clear supernatant, avoiding the fluffy layer over the pellet, to yield the final S-100 fraction.e) Freeze in aliquots, in liquid nitrogen and store at -80?C
