蛋白抽提Protocol(全)lyz解析

Loading Buffer煮细胞抽提总蛋白Protocol

1. 细胞计数（约1×106的细胞），离心收集细胞（2000rpm×5min）；

2. 预冷1×PBS 500ul重悬洗一次，转至500ul EP管（2000rpm×5min）；

3. 去上清，后甩一次，将上清去尽；

4. 按每1×106的细胞加50ul loading buffer 加入2×loading；

5. Vortex一下，煮10～15min一次×2～3次直至无粘丝；

6. 每次煮好将其放于冰上，静置约2min，Vortex一下，再甩一下；

7. 三次后用吸起，如没有粘丝，即可；

8. 每次上样约5ul（50ug/ul蛋白）

RIPA裂解抽提总蛋白Protocol

1. 细胞计数（约1×107的细胞），离心收集细胞（2000rpm×5min）；

2. 预冷1×PBS 1ml重悬洗2次，转至1.5ml EP管（2000rpm×5min）；

3. 去上清，后甩一次，将上清去尽；

4. 按照如下比例加入裂解试剂：

RIPA ： 300ul/1×107细胞

PMSF ： 3ul(1:100)

Cocktail ： 0.3ul(1:1000)

5. 吹打均匀，冰上放置30-40min，中间每10分钟Vortex一次；

6. 4℃预冷离心：10000rpm ×10min；

7. 收集上清，转移至已预冷新EP管，即为总蛋白。

【为了浓缩蛋白，加RIPA的量改为200ul或者更低，可稍微延长冰上裂解时间，而RIPA（1）＋PMSF（1：100）＋Cocktail（1：1000）的比例不变 】

核，浆蛋白抽提Protocol

一. 收核-浆蛋白

1.

2.

3.

4.

收浆蛋白

细胞计数（约1.5×107的细胞），离心收集细胞（1100rpm×5min）；

用4℃预冷的PBS重悬，转至1.5mL的EP管中，离心（4000rpm×5min，4℃）；

去上清，加入A Buffer 1mL/1×107 cell，重悬，4℃放置10min，离心（2500rpm×3min，4℃）；

去上清，加入A’ Buffer 250uL/1.5×107 cell，重悬，4℃放置3min，离心（5000rpm×1min，4℃），吸取上清（上清是浆蛋白）；

收核蛋白

再用A’ Buffer 500uL/1.5×107 cell，重悬，4℃放置3min，离心（5000rpm×1min，4℃），吸走上清，12000rpm×30sec，把上清完全吸干净；

剩余沉淀中加入B’ Buffer (或者RAPI)50uL，重悬（振荡），4℃放置40min（每隔10min振荡一次）；

离心（12000×10min，4℃），取上清即为核蛋白，－80℃保存。

5.

6.

7.

Buffer A的配制：

终浓度 母液

Hepes PH 7.9 10mM 1M

MgCl2 1.5mM 2M

KCl 10mM 250mM

DTT 0.5mM 0.1M

剩余体积用ddH2O补足至要求体积!

Buffer A’的配制：

配40Ml (10ml)溶液所需的量

400uL (100 ul)

30uL (7.5 ul)

1.6mL (400 ul)

200uL (50 ul)

Buffer A’(2ml) = 1960ul Buffer A + 40uL 10%NP-40 + 20uL 10mg/mLPMSF + 1uL Aprotinin

Buffer A’(1ml) = 980ul Buffer A + 20uL 10%NP-40 + 10uL 10mg/mLPMSF + 1uL Aprotinin

（Aprotinin可以用cocktail代替,而且只要核蛋白的时候可以不加）

Buffer B 的配制：

终浓度

Hepes PH 7.9 20mM

25%

甘油

NaCl 0.42M

EDTA 0.2mM

DTT 0.5mM

NP-40 0.2%

剩余体积用ddH2O补足至要求体积!

Buffer B’的配制：

母液

1M

50％

3M

0.5M

0.1M

10％

配40mL(10ml)溶液所需的量

800uL (200 uL)

20mL (5 mL)

5.6mL ( 1.4 mL)

16uL ( 4 uL)

200uL ( 50 uL)

800uL ( 200 uL)

Buffer B’ = 1mL Buffer B + 10uL 10mg/mLPMSF + 0.5uL Aprotinin(可以用cockltail代替)

核基质蛋白抽提Protocol

1、 细胞计数（约1×107的细胞），离心（1100rpm×5min）收集细胞；

2、 用4℃预冷的PBS重悬，将细胞移至1.5ml EP管中，1×PBS洗一遍；

3、 加300ul RIPA/1×107的细胞，裂解细胞，至冰上15-30min；

RIPA＋PMSF（1：100）＋Cocktail（1：1000）

4、 4℃预冷离心：13000rpm ×10-15min；

5、 将离心后的上清转移至新的已4℃预冷的1.5ml EP管，冰上备用(总蛋白)；

6、 用１×PBS洗管底的pellet×2遍；

7、 加入Urea-Lysis buffer 10-15ul裂解pellets，vortex 10min；

8、 4℃预冷离心：13000rpm ×10min；用BufferA 90ul稀释，此为核基质蛋白。

RIPA（PH 8.0）：

150mM NaCL

1% NP-40

0.5% DOC

0.1% SDS

50mM Tris

Urea-Lysis buffer（PH 8.0）：

100mM NaH2PO4

10mM Tris-HCL

300mM NaCL

8M urea

Buffer A：

100mM NaH2PO4

10mM Tris-HCL

300mM NaCL

1% Triton X-100

cytoplasmic cell fractions were prepared by incubating the cells in icecold

Iso-Hi buffer

140 mM NaCl

25 mM Tris [pH 7.4]

1.5 mM MgCl2)

0.5% Nonidet P-40

for 5 min and then subjecting them to low-speed centrifugation to collect the nuclei. Nuclei were

incubated in

high-salt extraction buffer

20 mM HEPES (pH 7.9)

25% (v/v) glycerol

0.5 MNaCl

1.5 mM MgCl2

0.2 mM EDTA

0.5 mM phenylmethylsulfonyl fluoride (PMSF)

0.5 mM DTT;

for 1 h at 4C. The DNA-particulate fraction was pelleted by microcentrifugation (15,000 rpm),

washed once in high-salt buffer, solubilized in radioimmunoprecipitation assay buffer(RIPA), and

sonicated to sheer the DNA. Equal amounts of all fractions were precipitated with trichloroacetic

acid, resuspended in protein sample buffer, and separated on a sodium dodecyl sulfate (SDS)–10%

polyacrylamide gel.

Accurate transcription initiation by RNA polymerase II in a soluble extract from

isolated (nuclear extraction)

buffer A :

10 mM HEPES (pH 7.9 at 4C)

1.5 mM MgCl2

10 mM KC1

0.5 mM DTT;

buffer B

0.3 M HEPES (pH 7.9)

1.4 M KC1

0.03 M MgCl2;

buffer C

20 mM HEPES (pH 7.9)

25% (v/v) glycerol

0.42 MNaCl

1.5 mM MgCl2

0.2 mM EDTA

0.5 mM phenylmethylsulfonyl fluoride (PMSF)

0.5 mM DTT;

buffer D

20 mM HEPES (pH 7.9)

20% (v/v) glycerol,

0.1M KCl,

0.2 mM EDTA

0.5 mM PMSF

0.5 mM DTT

DTT and PMSF were added fresh to the buffers just before use.

1，Pelleted cells were then suspended in five volumes of 4C phosphate buffered saline and

collected by centrifugation as detailed above; subsequent steps were performed at 4°C.

2，The cells were suspended in five packed cell pellet volumes of buffer A and allowed to stand

for 10 min.

3，The cells were collected by centrifugation as before and suspended in two packed cell pellet

volumes (volume prior to the initial wash with buffer A) of buffer A and lysed by 10 strokes of a

Kontes all glass Dounce homogenizer (B type pestle).

The homogenate was checked microscopically for cell lysis and centrifuged for 10 min at 2000

rpm in a Sorvall HG4L rotor to pellet nuclei. The pellet obtained from the low speed

centrifugation of the homogenate was subjected to a second centrifugation for 20 min at 25,000 ga

(Sorvall SS34 rotor), to remove residual cytoplasmic material an d this pellet was designated as

crude nuclei. These crude nuclei were resuspended in 3 ml of buffer C per 109 cells with a Kontes

all glass Dounce homogenizer (10 strokes with a type B pestle). The resulting suspension was

stirred gently with a magnetic stirring bar for 30 min and then centrifuged for 30 min at 25,000 g

(Sorval SS34 rotor). The resulting clear supernatant was dialyzed against 50 volumes of buffer D

for five hours. The dialysate was centrifuged at 25,000 g (Sorvall SS34 rotor) for 20 min and the

resulting precipitate discarded. The supernatant, designated the nuclear extract, was frozen as

aliquots in liquid nitrogen and stored at -80°. The protein concentration was usually 6 to 8 mg per

ml and 15 to 20 mg of protein were obtained from 109 cells.