5种蛋白双向电泳总方案

1、蛋白质双向电泳总方案一、植物蛋白的提取1 chloroform/acetone method: this method was performed according to the protocol described by xie et al. (2007), with modificatio ns. the modificatio ns in eluded differe nt tissue amount, reage nt volume, and incubati on time. these modificati ons also applied to the other three

2、methods described below.about 0.5 g of leaf or 1.5 g of root sample was homogenized in 4.0 ml of extraction buffer (0.1 m kci, 0.5 m tris-base at ph 7.5, 0.05 m edta, and 2% 2-mercaptoethanol) for 10 min. the mixture was shaken on ice for 2 h and centrifuged at 12,000 g for 15 min at 4°c the su

3、pernatant was transferred to a new tube, and an equal volume of water saturated chloroform and isoamyl alcohol (24/1, v/v) was added the tube was shaken at 4°c for 30 min and then stored on ice for 10 min before centrifugation at 10,000 g for 10 min at 4°c. the upper and bottom phases were

4、 removed. then 4 ml of water was added to the in terphase together with an equal volume of water-saturated chloroform and isoamyl alcohol (24/1, v/v); the mixture was homogenized, allowed to stand on ice for 10 min, and then cen trifuged at 10,000 g for 10 min at 4°c. the treatme nt was repeate

5、d twice the in terphase was washed with ice-cold acet one three times the final pellets were vacuum-dried and dissolved in resolubilization solution (8 m urea, 2 m thiourea, 2% chaps, 1% dithiothreitol dtt, 1% pharmalyte).2 phenol/ammonium acetate method: this method was developed following the prot

6、ocol described by hurkman and tanaka (1986), with mod讦ication. about 0.5 g of leaf or 1.5 g of root sample was homogenized in 4.0 ml extraction buffer (50% phenol, 0.45 m sucrose, 25 mm edta, 1% v/v 2-mercaptoethanol, 250 mm tris-base, ph 8.8) for 10 min. samples were transferred to phenolresistant

7、screw-cap tubes and incubated on a mixer for 30 min at 4°c and then centr讦uged at 5000 g for 10 min at 4°c the upper phenol-phase was removed and added to five volumes of ice-cold 0.1 m ammonium acetate and 1% 2-mercaptoethanol in 100% methanol and then mixed before placing at - 20°c

8、for 2 h. precipitated protein was collectedby 10 min centrifugation at 12,000 g. pellets were thoroughly washed twice with 20 ml of 0.1 m amm onium acetate in 100% metha nol, followed by two washes with ice-cold 80% acetone,a nd a final wash with ice-cold 70% ethanol. the final pellets were vacuum-d

9、ried and dissolved in resolubilization solution.3 tfis-base/acetone method: this protocol was performed according to rabilloud(1998), with modifi cation about 0.5g of leaf or 1.5 g of root sample was homogenized in 4.0 ml of extraction buffer (40 mm tris-base, 5 m urea, 2 m thiourea,2% w/v chaps, 5%

10、 w/v polyvinyipyrollidone, and 2% 2mercaptoethanol). the homogenate was centr讦uged for 15 min at 12,000 g. the protein in the supernatant was precipitated by adding four volumes of ice-cold acetone containing 0.07% (w/v) 2-mercaptoethanol, incubated at - 20°c for at least 2 h, and then cen trif

11、uged for 15 min at 12,000 g. the pellet was washed with ice-cold acet one containing 0.07%2-mercaptoethanol, incubated at 一 20°c for 2 h, and centrifuged again at4°c the washing was repeated twice the final pellets were vacuum-dried and dissolved in resolubilizationsoluti on.4 tca/acetone

12、method: this protocol was modified from the method described bydamerval et al. (1986). about 0.5 g of leaf or 1.5 g of root sample was homogenized for 10 min and incubated with 10 ml of precipitation solution (10% tca and 0.07% 2-mercaptoethanol in acet one) for 2 h at-20°c the precipitated pro

13、tei ns were pelleted and washed with ice-cold acet one containing 0.07% 2-mercaptoetha nol to remove pigme nts and lipids until the supernatant was colorless the pellet was vacuum-dried, resuspended in resolubilization solution,and son icated to extract protei ns. in soluble tissue was removed by ce

14、ntrifugatio n at 21,000 g for 20 min.5 mg/np-40： two grams of plant tissues were placed in liquid nitrogen and then stored at -80'c.the plant tissue was transferred to a prechilled mortar, and ground with a pestle in liquid nitrogen to a fine powder. the powder was homogenized in 10 ml of ice-co

15、ld mg/np-40 extraction buffer containing 0.5 m tris-hci, ph 8.3, 2% v/v np-40, 20 mm mgch, 2% v/v p-mercaptoethanol, 1 him phenylmethylsulfonylfluoride (pmsf) and 1% w/v polyvinylpolypyrrolidone(pvpp). after centrifugation at 12000g for 15 min at 4°c, proteins in the supernatant were precipitat

16、ed by adding four volumes of cold acetone at -20 °c for 30 min for analysis of total protein by 2-de.注：除tca/acetone外其它方法均为可溶性蛋白提収方法，在实验过程中发现用protein extraction buffer提取后的蛋白液用预冷的tca/acetone含0.07%卩-mercaptoethanol沉淀效果 比纯丙酮要好！因此，可以考虑其它蛋白提取方案与tca/acetone结合。二、蛋白的裂解1裂解液的配方©8 mol/l urea,2 mol/l t

17、hiourea,4 % chaps, 1 % np-40, 4 % tritonx-100,65 mmol/l dtt,0.5 mmol/l pmsf,0.5 % phannalyte 3-10 8 mol/l urea, 2 mol/l thiourea, 4% chaps, 20 mmol/l trisbasc, 30 mmol/l dte, 2% pharmalyte ph 3-10.®8murea,2m thiourea, 2%3-(3-cholamidopropyl)dimethylamonio-l -propanesulfonatechaps, 1% dithiothre

18、itol dtt, 1% pharmalyte2 m thiourea, 7m urea, 4% (w/v) chaps, 1% (w/v) dtt, 2%(v/v) carrier ampholytes (ph3-10)注：以上仅供参考，通常用的一个配方是：2.1g urea, 0.76gthiaurea, 0.05g dtt, 0.2gchaps, 25ul carrier ampholytes (ph3-10)最后定容至5ml.2蛋白的裂解称取蛋白质干粉按10-30ul/mg的比例加入裂解液，用枪头混匀后在冰浴超声促溶30- 40 min, 12000r/min离心取上清用于测定蛋白浓度

19、。注：裂解液的加入比例根据样品蛋白浓度决定，上样体积为40-80ui都很好！其实只要能上 到管内都是可以的！3蛋白含量的测定bradford法 标准曲线滴定将10支干燥洁净的试管编号，按下表加入试剂摇匀，室温放置半分钟后各管加入5mlbradford i作液摇匀，室温放置5分钟后，一 0号管为空白调零测定，其余各管595nm 出相对吸光度管号0123456789ddh2o/ul100908070605040302010蛋白质标准 液(ul)0102030405060708090裂解液/ul1010101011010101010100.1m hcl/ul10101010101010101010

20、bradford储存液350mg 考斯亮蓝，g-250 4-100ml95%的乙醇+250ml85%(u/l)磷酸 bradford工作液 500ml 量：425mlddh2o,15ml5%的乙醇,30ml85%(u/l)磷酸,30ml bradford 储存液 100ml 量：85.15ml dd h20,2.85ml100%乙醇,6ml85%(u/l)磷酸,6ml bradford 储存液 200ml i作液：170.3mldd h2o,6ml95%乙醇,12ml85%(u/l)磷酸,12ml 储存液蛋白质标准液：加10mgbsa标准液品溶于10ml水，配置1 mg/ml bsa蛋白溶液

21、样品溶度的测定：在干燥洁净的试管中依次加入90ul dd h20.将测定蛋白样品10ul0.1m hcl10ul混匀，加入5ml工作液摇匀，室温放置5分钟后，测定其在595nm出相对吸光 度，照标准曲线读取浓度。三、蛋白双向第一向等电聚焦(ief)1. 一向管的清洗用重侪酸钾洗液(重侪酸钾15g, ddh2o 30ml,浓硫酸300ml)超声清洗，后用双蒸憾洗 至管内清洁。2 一向胶的配置(17cm)28.38% acr+1.62%bis制胶4根5根6根7根8根9根10根urea/g0.961.201.441.681.922.162.4010%np40/ml0.400.500.600.700.

22、800.901.0015%acr/bis(ml)0.2330.290.350.410.4670.5230.58ddh20/ml0.5670.710.850.9911.1331.2771.42两性电解质 ph (3. 5-10)/ul253037.5043.755056.2562.5ph(5-8)/ul75120112.50131.25150168.75187.510%ap/ul3.744.675.606.547.488.49.3temed/ul0.9331.171.401.631.8662.1032.3注：两性电解质的比例(310： 5-8)可以根据自己的样品调，表中的为1： 3,用1： 4的

23、 效果更好！大口径的一向管六根量能灌足4根，小管5根量能灌4根。夏天温度比较高时 temed可以适当减三分一的量。10%ap和temed都是最后要灌胶时再加。加完尿素后 可以冰浴超声溶解下。 灌胶把胶吸至17.5cm处，上端用水封，待其凝固，约1小时 上样用微量进样器进样，或者当上样体积较大时(大于40ul)可用移液器上样。 加入覆盖液覆盖液配方(np-40 2ml ,am (3.5-10)1 ooul, p-me 500ul ,urea 4.8g)然后定容至 10ml 覆盖液的量不一定要固定的量20ul差不多了。 上下槽缓冲液上槽:naoh 20mm 0. 4g-500ml下槽：磷酸 10m

24、m 0.33711)1 5001111 电泳程序设定步骤1231200v 30min200v 30min200v 30min2300v 30min300v 30min300v 30min3400 v 30min400v 30min400v 30min4500 v 30min500v 30min500v 30min5600 v 30min600v 30min600v 30min6800 v 16.5h800v14h800v16h71000 v 4h1000v 4h1000v 2h81200v 2h1200v 2h电泳结束后用注射器将胶条挤出至装有第二向电泳缓冲液的培养皿内，后将胶条放入10ml

25、离心管，4°c保存约5min.3胶条的平衡每根胶条加入5ml平衡液配方如下，平衡20min平衡液200ml100ml500ml60mmtris-hcl(ph6.8)8ml4ml20ml2%sds4g2g10g5% p -me10ml5ml25ml10%甘油20ml10ml50ml0.05%澳酚蓝o.lg0.05g0.25g四蛋白电泳第二向(sds-page)30%丙烯酰胺储液的配置(30%acr, 0.8%bis)分离胶的配置以下是每板胶的配置，根据自己的需要选择胶浓度，植物的话叶片一般为12.5%,根为10%, 最好做下预实验选择胶浓度！药品/浓度5%7.5%10%12.5%15%丙烯酰胺6.7ml10ml13.3ml16.7ml20ml1.5mtris-hcl(ph8.8)10ml10ml10ml10ml10ml10%sds0.4ml0.4ml0.4ml0.4ml0.4mlddh2o22.7ml19.4ml16.1ml12.8ml9.5ml10%ap200ul200ul200ul200ul200ultemed13.3ul1