蛋白质的十种提取方法

，

0.5%DTT(

硫苏糖醇

)

，

2% Ampholine (Amersham Pharmacia Biotech Inc

，

pH3.5- 10)

，

6% Triton X-100]

< br>，

37

℃温

育

< br>30min

，期间搅

动几次，

28

度

（温度低，高浓度的尿素会让溶液结冰）

16000 r/min

离心

15 min

，离心力

越大时间长一点

越好！上清即可上样电泳。或者

-70

度保存

七、

植物根中蛋白质的抽取（

phenol

）

(1) sample,

液氮研磨

(2)

装

1.5 ml centrifuge

用

tube

(3)

加

1M KH2PO4+K2HPO4 700 ul

(4) 12000 rpm, 4

度

, 10-15minite

(5)

取上层液，蛋白质就在里面

八、

SDS

extraction

followed

by

acetone

precipitation

–

simple

extraction

protocol

that

does not require phenol.

Recommended start protocol for whole tissue extractions.

（

hg p

）

1.

Grind

1

g

of

fresh

tissue

to

a

powder

with

liquid

nitrogen

in

a

mortar

and

pestle.

2.

Add

5

mL

of

extraction

media

(0.175

M

Tris- HCl,

pH

8.8,

5%

SDS,

15%

glycerol,

0.3

M DTT) directly to

mortar and continue grinding for an additional 30 sec.

3.

Filter

homogenate

through

two

layers

of

miracloth

into

a

50

mL

Falcon

tube

at

room

temperature.

4. Immediately add 4 volumes of ice cold 100% acetone to filtered homogenate, mix

by vortexing and place at -20

C for at least one hour to precipitate proteins.

5.

Centrifuge

at

5000

g

for

15

min

to

collect

precipitated

protein,

decant

supernatant.

6. Gently blot residual

acetone from

container with Kimwipe and then wash pellet

in

15-20 mL of cold 80%

acetone.

Be

sure

to

thoroughly

break-up

pellet

by

pipetting,

vortexing

or

sonication.

7. Repeat steps 5 and 6.

8. Collect final protein precipitate by centrifugation at 5000 g for 15 min and dry

pellet by inverting on Kimwipe

for 15 min at 37 C.

9.

Resuspend

final

pellet

in

0.5-1

mL

of

IEF

extraction

solution

(8

M

urea,

2

M

thiourea,

2% CHAPS, 2% Triton

X-100, 50 mM DTT, 0.2% pH 3-10 ampholytes) by pipetting and vortexing at 25-30 C.

Incubate sample for 1 h at

room

temperature

with

agitation.

Do

not

heat

sample

under

any

circumstances

as

this

will lead to carbamylation of

proteins.

10. Centrifuge for 10 min at 12000 g and use supernatant to rehydrate IPG strips.

11. If protein quantitation is necessary, precipitate protein sample with TCA or

acetone prior to performing

Bradford

or

Lowry

assay

as

detergents

and

reducing

agents

interfere

with

these

assays.

Phenol

extraction

followed

by

methanolic

ammonium

acetate

precipitation

–

an

effective protocol for sample

preparation

from

protein-poor,

recalcitrant

tissues

such

as

plants

(see

Hurkman

and

Tanaka, 1986, Plant

Physiology 81:802-80

九、

材料：细菌蛋白（

puc18

）

用甲醇提取 的，

冻干后用缓冲液溶解的。

样品缓冲液是一般的。

2%

的

SDS< /p>

，

20mmol

的

2-

巯基乙

醇。

十、

线粒体蛋白的提取

(bioon)

Isolation for Mitochondria

Modification by Bioon

Materials and reagents:

homogenizing buffer:

100 mM mannitol

10 mM Tris-HCl buffer (pH 7.5)

5 mM MgCl2

关

于

蛋

白

质

1 mM EGTA

1 mM DTT

leupeptin (0.1 ug/ml)

0.1M Na2CO3

Methods:

- 10*6 Cells were washed with ice-cold PBS and lysed by homogenizing in 1 ml buffer

(ice-cold) containing 100

mM mannitol, 10 mM Tris, 5 mM MgCl2, 1 mM EGTA, 1 mM DTT, leupeptin (0.1 ug/ml)

- Subjected to Polytron homogenization for three-four bursts of 3-10 s each at a

setting of 6.5.

-

Intact

cells

and

nuclei

were

separated

by

centrifugation

at

120

g

for

5

min

at

4

℃

-

Supernatants

were

centrifuged

at

10,000

g

for

10

min

to

collect

the

heavy

(mitochondrial) membrane pellet.

-

Cytoplasmic

fractions

were

obtained

by

centrifuging

supernatants

at

100,000

g

for

30 min.

- Resuspended pellet to 0.25mg/ml in fresh preparation of 0.1M Na2CO3 (pH 11.5)

- Incubated on ice for 30 min.

- Ultracentrifugation at 100000g for 1h at 4

℃

to precipitate the mitochondria

membrane protein. And the

supernatants are mitochondrial matrix. 0.5mg of proteins in mitochondria can get

100ug of proteins (the

alkali-resistant fractions)

Ref.: PNAS, 2002

，

99

：

12825

–

< br>12830

本方法只适用于提大鼠细胞线粒体蛋白，而不适用于线粒体功能检测

考

亮

马

蓝

斯

染

色

一

产

介：

品

简

本

考

马

斯

亮

蓝

染

色

试

剂盒

(Comm

assie

Blue

Staining

Kit)

采

最

的

斯

用

经

考

亮

了

典

马

蓝

< br>染色

和

方法，

以

脱

色

考

马

亮蓝

R250

为

斯

染

料，

可用于

SDS- PA

GE

或

非

变

性

P

AGE

等

蛋

白

电泳凝

胶

规

和

常

色< /p>

脱

的

染

色，或< /p>

Western

转膜后

P

AGE

胶

上

残

余蛋白

的

测。

检

使

用

本

试

盒< /p>

用

，

剂

采

常

色

方

规

脱

法

时

染

色

累

间

达到

2-3

小时

后

可< /p>

以

观

察

到

蛋

带；

采用

白

条

快