The choice of the lysis buffer depends on the protein investigated. Where the target protein is a soluble protein or tightly bound to cytoskeletal elements, a soft or strong extraction buffer is required. As soon as lysis occur, proteolysis, dephosphorylation and denaturation begins. These events can be slowed down tremendously if samples are kept on ice or at 4ºC at all times and appropriate inhibitors are included in the extraction buffer.
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 4ºC 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 -80ºC.
Nuclear Protein Extraction:
a) Prepare buffer A (10 mM HEPES; 1.5 mM MgCl2; 10 mM KCl; 0.5 mM DTT; 0.05% NP-40 pH 7.9) and buffer B (5 mM HEPES; 1.5 mM MgCl2; 0.2 mM EDTA; 0.5 mM DTT; 26% Glycerol pH 7.9) and store on ice.
b) Add around 500 µl of buffer A per large Petri dish on ice and scrape thoroughly, leave on ice for 10 minutes.
c) Centrifuge at 4ºC at 3000 rpm for 10 minutes.
d) Remove supernatant and keep it - this contains everything except large plasma membrane pieces, DNA, nucleoli). Use 10 µl for protein quantification.
e) Ressupend pellet on ice in 374 µl of buffer B and add 300 mM NaCl.
f) Homogenize in a glass homogenizer on ice. Leave on ice for 30 minutes.
g) Centrifuge at 24,000 g for 20 minutes at 4ºC.
h) Alliquot supernatant, remove 10 µl for protein quantification and store at -80ºC.
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