1、 Loading Buffer 煮细胞抽提总蛋白 Protocol1. 细胞计数（约 1106 的细胞） ，离心收集细胞（2000rpm 5min） ；2. 预冷 1PBS 500ul 重悬洗一次，转至 500ul EP 管（2000rpm5min） ；3. 去上清，后甩一次，将上清去尽；4. 按每 1106 的细胞加 50ul loading buffer 加入 2loading；5. Vortex 一下，煮 10 15min 一次23 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. 三次后用枪吸起，如没有粘丝，即可；8. 每次上样约 5ul（
2、50ug/ul 蛋白）RIPA 裂解抽提总蛋白 Protocol1. 细胞计数（约 1107 的细胞） ，离心收集细胞（2000rpm 5min） ；2. 预冷 1PBS 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/1107 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
3、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-浆蛋白收浆蛋白1. 细胞计数（约 1.5107 的细胞） ，离心收集细胞（1100rpm 5min） ；2. 用 4预冷的 PBS 重悬，转至 1.5mL 的 EP 管中，离心（4000rpm5min，4） ；3. 去上清，加入 A Buffer 1mL/1107 cell，重悬，4放置 10min，离心（2500rpm3min，4） ；4. 去上清
4、，加入 A Buffer 250uL/1.5107 cell，重悬，4放置 3min，离心（5000rpm1min，4） ，吸取上清（上清是浆蛋白） ；收核蛋白5. 再用 A Buffer 500uL/1.5107 cell，重悬，4放置 3min，离心（5000rpm1min，4） ，吸走上清，12000rpm30sec，把上清完全吸干净；6. 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） ；7. 离心（1200010min，4） ，取上清即为核蛋白，80保存。Buffer A 的配制：终浓度 母液 配 40Ml
5、 (10ml)溶液所需的量Hepes PH 7.9 10mM 1M 400uL (100 ul)MgCl2 1.5mM 2M 30uL (7.5 ul)KCl 10mM 250mM 1.6mL (400 ul)DTT 0.5mM 0.1M 200uL (50 ul)剩余体积用 ddH2O 补足至要求体积!Buffer A的配制：Buffer A(2ml) = 1960ul Buffer A + 40uL 10%NP-40 + 20uL 10mg/mLPMSF + 1uL AprotininBuffer A(1ml) = 980ul Buffer A + 20uL 10%NP-40 + 10uL
6、 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 母液 配 40mL(10ml)溶液所需的量Hepes PH 7.9 20mM 1M 800uL (200 uL)甘油 25% 50 20mL (5 mL)NaCl 0.42M 3M 5.6mL ( 1.4 mL)EDTA 0.2mM 0.5M 16uL ( 4 uL)DTT 0.5mM 0.1M 200uL ( 50 uL)NP-40 0.2% 10 800uL ( 200 uL)剩余体积用 ddH2O 补足至要求体积!B
7、uffer B的配制：Buffer B = 1mL Buffer B + 10uL 10mg/mLPMSF + 0.5uL Aprotinin(可以用 cockltail 代替)核基质蛋白抽提 Protocol1、 细胞计数（约 1107 的细胞） ，离心（1100rpm5min）收集细胞；2、 用 4预冷的 PBS 重悬，将细胞移至 1.5ml EP 管中， 1PBS 洗一遍；3、 加 300ul RIPA/1107 的细胞，裂解细胞，至冰上 15-30min；RIPAPMSF（1：100）Cocktail（1：1000）4、 4预冷离心：13000rpm 10-15min；5、 将离心后的
8、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PBS 洗管底的 pellet2 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 NaCL1% NP-400.5% DOC0.1% SDS50mM TrisUrea-Lysis buffer（PH 8.0）：100mM NaH2PO410mM Tris-HCL300mM NaCL8M ureaBuffer A：1
9、00mM 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 collect the nuclei. Nuclei were incub
10、ated 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 in high-salt buffer, solubilized in
11、 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 R
12、NA 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 MgCl20.2 mM EDTA0.5 mM phenylmethylsulfonyl
13、 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 and collected by centrifugation as de
14、tailed above; subsequent steps were performed at 4C. 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 wit
15、h 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 o
16、f 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 homoge
17、nizer (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
18、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.
