| Protocol of the protein extraction method | | 点击： 作者：51protocol收集 来源： 时间： 2007-06-02 本站论坛 |
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Protocol of the protein extraction method
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
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