Protocol of the protein extraction method
互联网
1. One-step extraction method:
在冰上将组织在匀浆缓冲液中（25mM NaH
2
PO
4
pH7.2，1.5mM EDTA，10% Glycerol，10mM Na
2
MO
4
，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® or power homogenizer (Polytron, or Tekmar"s TISSUMIZER® or equivalent). The sample volume should not exceed 10% of the volume of TRIZOL Reagent used for homogenization.
b. Cells Grown in Monolayer
Lyse cells directly in a culture dish by adding 1 ml of TRIZOL Reagent to a 3.5 cm diameter dish, and passing the cell lysate several times through a pipette. The amount of TRIZOL Reagent added is based on the area of the culture dish (1 ml per 10 cm 2) and not on the number of cells present. An insufficient amount of TRIZOL Reagent may result in contamination of the isolated RNA with DNA.
