蛋白抽提Protocol(全)lyz.doc

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（2000rpm5min）；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1015min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（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（2000rpm5min）；2. 预冷1PBS 1ml重悬洗2次，转至1.5ml EP管（2000rpm5min）；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（1100rpm5min）；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，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 (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 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 7.920mM1M800uL (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)剩余体积用ddH2O补足至要求体积!Buffer 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：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 collect 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 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 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 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 detailed 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 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