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1) The sample was grinded to power with liquid nitrogen;
2) Add 10mL cooled acetone contained 10% TCA to 1g sample power at  -20¡ãC for 1 hour;
3) Centrifuge by 15000g for 15 min at  4¡ãC, collect the  deposit  and  add cooled  acetone at -20¡ãC for 1 hour;
4) Repeated the step 3;
5) Centrifuge  by  15000g  for  15  min  at  4¡ãC,  collect  the  deposit  and  dried  by vacuum freezed dryer;
6) Suspends the deposit in cold phenol extraction buffer;
7) Add an equal volume of  phenol saturated with Tris-HCl  (pH  7.5) and shake  the mixture for 30 min at 4 ¡æ;
8) Centrifuge at 5000g for 30 min at 4 ¡æ and collect the upper phenolic phase;
9) Add  equal  phenol  extraction  buffer  to  the  collected  phenolic  phase,  repeat steps 2-3 and collect the upper phenolic phase again;
10) Add 5 volumes of cold 0.1 M ammonium acetate in methanol to the collected phenol phase, store at ¨C20 ¡æ for 1 hour;
11) Centrifuge the sample for 30 min at 5000g at 4 ¡æ;
12) Add 2 volumes (based on the volume of the last collected phenolic phase) ice-cold methanol to wash the pellet, mix gently;
13) Centrifuge the sample for 30 min at 5000g at 4 ¡æ;
14) Repeat Steps 12and 13 two more times;
15) Repeat Steps 12 and 13 two more times using acetone instead of methanol;
16) Centrifuge the sample for 30 min at 5000g at 4 ¡æ, dry the deposit;
17) The deposit was dissolved in lysis solution at 30¡ãC for 1 hour;
18) Centrifuge the solution by 15000g for 15 min at room temperature, collect the supernatant and centrifuge again;
19) The supernatant was the extracted protein solution.

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