Protocol of the protein extraction method

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1.  One-step extraction method:
在冰上将组织在匀浆缓冲液中（25mM NaH2PO4 pH7.2，1.5mM EDTA，10% Glycerol，10mM Na2MO4，10mM NaF，2μM Aprotinin，5μM Leupeptin，2mM PMSF）剪碎，用高速搅拌器匀浆，而后于10,5000g 超离心一小时。取上清，用Brandford法定蛋白浓度[116]，分装冻存于－20℃。抽提的组织蛋白用于Western blot。

2.  TCA method:
(1)  Homogenization in 1ml lysis bufferⅠ;
(2)  Centrifugate the lysate at 13,000 rpm×10 min in 4℃;
(3)  Transfer the supernatant to a new ep tube, add ice-cold 10% TCA and store on ice for more than 30 min;
(4)  Centrifugate at 13,000 rpm×10 min in 4℃;
(5)  Discard the supernatant and wash the pellet once by ice-cold acetone;
(6)  Centrifugate at 13,000 rpm×10 min in 4℃;
(7)  Dissolve the pellet in lysis bufferⅡ, and break it up to dissolve completely by ultrasonic;
(8)  Centrifugate at 13,000 rpm×10 min in 4℃;
(9)  Dispart the supernatant, measure the concentration of the protein.

Annotation: the component of buffer:
Lysis bufferⅠ (250ml):
   Glycerd:    25ml (10%)
  Triton X-100:    2.5ml (1%)
  Hepes (PH7.5):    12.5ml (50mM)
   NaCl (150mM):   37.5ml
   NaF (2mM)
  EDTA (PH8.0 2mM)   500μl
   ddH2O    172.5ml

Lysis bufferⅡ
   Urea     7M
   Thioncea    2M
   CHAP     4%
   Tris     40mM
   SDS     0.1%

1.  Trizol method (invitrogen):

(1). HOMOGENIZATION (see notes 1-3)

a. Tissues

Homogenize tissue samples in 1 ml of TRIZOL Reagent per 50-100 mg of tissue using a glass-Teflon