蛋白抽提Protocol(全)lyz

1、 Loading Buffer煮細(xì)胞抽提總蛋白Protocol1. 細(xì)胞計(jì)數(shù)（約1×106的細(xì)胞），離心收集細(xì)胞（2000rpm×5min）；2. 預(yù)冷1×PBS 500ul重懸洗一次，轉(zhuǎn)至500ul EP管（2000rpm×5min）；3. 去上清，后甩一次，將上清去盡；4. 按每1×106的細(xì)胞加50ul loading buffer 加入2×loading；5. Vortex一下，煮1015min一次×23次直至無粘絲；6. 每次煮好將其放于冰上，靜置約2min，Vortex一下，再甩一下；7. 三次后用槍吸起，如沒有

2、粘絲，即可；8. 每次上樣約5ul（50ug/ul蛋白）RIPA裂解抽提總蛋白Protocol1. 細(xì)胞計(jì)數(shù)（約1×107的細(xì)胞），離心收集細(xì)胞（2000rpm×5min）；2. 預(yù)冷1×PBS 1ml重懸洗2次，轉(zhuǎn)至1.5ml EP管（2000rpm×5min）；3. 去上清，后甩一次，將上清去盡；4. 按照如下比例加入裂解試劑：RIPA ： 300ul/1×107細(xì)胞PMSF ： 3ul(1:100)Cocktail ： 0.3ul(1:1000)5. 吹打均勻，冰上放置30-40min，中間每10分鐘Vortex一次；6. 4預(yù)冷離心：1

3、0000rpm ×10min；7. 收集上清，轉(zhuǎn)移至已預(yù)冷新EP管，即為總蛋白。【為了濃縮蛋白，加RIPA的量改為200ul或者更低，可稍微延長冰上裂解時(shí)間，而RIPA（1）PMSF（1：100）Cocktail（1：1000）的比例不變 】核，漿蛋白抽提Protocol一. 收核-漿蛋白收漿蛋白1. 細(xì)胞計(jì)數(shù)（約1.5×107的細(xì)胞），離心收集細(xì)胞（1100rpm×5min）；2. 用4預(yù)冷的PBS重懸，轉(zhuǎn)至1.5mL的EP管中，離心（4000rpm×5min，4）；3. 去上清，加入A Buffer 1mL/1×107 cell，重懸，4放

4、置10min，離心（2500rpm×3min，4）；4. 去上清，加入A Buffer 250uL/1.5×107 cell，重懸，4放置3min，離心（5000rpm×1min，4），吸取上清（上清是漿蛋白）；收核蛋白5. 再用A Buffer 500uL/1.5×107 cell，重懸，4放置3min，離心（5000rpm×1min，4），吸走上清，12000rpm×30sec，把上清完全吸干凈；6. 剩余沉淀中加入B Buffer (或者RAPI)50uL，重懸（振蕩），4放置40min（每隔10min振蕩一次）；7. 離心（1

5、2000×10min，4），取上清即為核蛋白，80保存。Buffer A的配制：終濃度母液配40Ml (10ml)溶液所需的量Hepes PH 7.910mM1M400uL (100 ul)MgCl21.5mM2M30uL (7.5 ul)KCl10mM250mM1.6mL (400 ul)DTT0.5mM0.1M200uL (50 ul)剩余體積用ddH2O補(bǔ)足至要求體積!Buffer A的配制：Buffer A(2ml) = 1960ul Buffer A + 40uL 10%NP-40 + 20uL 10mg/mLPMSF + 1uL AprotininBuffer A(1ml

6、) = 980ul Buffer A + 20uL 10%NP-40 + 10uL 10mg/mLPMSF + 1uL Aprotinin（Aprotinin可以用cocktail代替,而且只要核蛋白的時(shí)候可以不加）Buffer B 的配制：終濃度母液配40mL(10ml)溶液所需的量Hepes PH 7.920mM1M800uL (200 uL)甘油25%5020mL (5 mL)NaCl0.42M3M5.6mL ( 1.4 mL)EDTA 0.2mM0.5M16uL ( 4 uL)DTT0.5mM0.1M200uL ( 50 uL)NP-400.2%10800uL ( 200 uL)剩余體

7、積用ddH2O補(bǔ)足至要求體積!Buffer B的配制：Buffer B = 1mL Buffer B + 10uL 10mg/mLPMSF + 0.5uL Aprotinin(可以用cockltail代替)核基質(zhì)蛋白抽提Protocol1、 細(xì)胞計(jì)數(shù)（約1×107的細(xì)胞），離心（1100rpm×5min）收集細(xì)胞；2、 用4預(yù)冷的PBS重懸，將細(xì)胞移至1.5ml EP管中，1×PBS洗一遍；3、 加300ul RIPA/1×107的細(xì)胞，裂解細(xì)胞，至冰上15-30min；RIPAPMSF（1：100）Cocktail（1：1000）4、 4預(yù)冷離心：13

8、000rpm ×10-15min；5、 將離心后的上清轉(zhuǎn)移至新的已4預(yù)冷的1.5ml EP管，冰上備用(總蛋白)；6、 用×PBS洗管底的pellet×2遍；7、 加入U(xiǎn)rea-Lysis buffer 10-15ul裂解pellets，vortex 10min；8、 4預(yù)冷離心：13000rpm ×10min；用BufferA 90ul稀釋，此為核基質(zhì)蛋白。RIPA（PH 8.0）：150mM NaCL1% NP-400.5% DOC0.1% SDS50mM TrisUrea-Lysis buffer（PH 8.0）：100mM NaH2PO410mM

9、 Tris-HCL300mM NaCL8M ureaBuffer A：100mM NaH2PO410mM Tris-HCL300mM NaCL1% Triton X-100cytoplasmic cell fractions were prepared by incubating the cells in icecoldIso-Hi buffer 140 mM NaCl25 mM Tris pH 7.41.5 mM MgCl2)0.5% Nonidet P-40for 5 min and then subjecting them to low-speed centrifugation to c

10、ollect the nuclei. Nuclei were incubated inhigh-salt extraction buffer 20 mM HEPES (pH 7.9)25% (v/v) glycerol0.5 MNaCl1.5 mM MgCl20.2 mM EDTA0.5 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

11、 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.Ac

12、curate 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 MgCl210 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) glycerol0.42 MNaCl1.5 mM MgCl20.

13、2 mM EDTA0.5 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 EDTA0.5 mM PMSF0.5 mM DTTDTT 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 a

14、nd 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 vol

15、umes (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 ob

16、tained 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 c

17、ells 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