【讨论分享帖】分享你做蛋白质组学实验的protocol

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标题:【讨论分享帖】分享你做蛋白质组学实验的protocol

米囡[使用道具]
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发表于 2012-12-3 14:27 资料个人空间短消息加为好友

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每个人在做自己蛋白质组学实验时都有一套对自己可行的实验方法，请大家把自己的实验验方法贴出来。希望这个是自己正在用的，并且能够得到不错结果的方法，而不是从网页上简单copy下来的。每个完整的protocol可以得到99个金钱的奖励。活动结束以后，将把所有的protocol集结成册，免费提供给大家。内容包括：蛋白质的提取双向电泳染色方法蛋白酶解质普鉴定更欢迎非2D的protocol。大家发贴时请注明您的实验材料和protocol的内容。比如说：植物蛋白质的提取方法 ...

米囡[使用道具]
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发表于 2012-12-3 14:28 资料个人空间短消息加为好友

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抛砖引玉，这是我以前发的老贴：植物蛋白质提取的三种protocols 1. TCA/丙酮法：（1）取4g果肉，用液氮在研钵中将其研磨成粉。（2）加入12.5%冰冷的TCA/丙酮（含0.07%β-巯基乙醇），匀浆液在-20 ºC下放置3h，纱布过滤。（3）滤液在20000g离心30min，收集沉淀。（4）用冷丙酮洗3次，沉淀在4ºC下干燥，备用。 2. 匀浆法：（1）称取4g果肉，用液氮在研钵中研磨成粉。（2）加入4ml的匀浆缓冲液(匀浆液中含有20 mM Tris-HCl (pH 7.5), 250 mM sucrose, 10 mM EGTA, 1 mM PMSF, 1 mM DTT, 以及1% Triton X-100)，继续研磨。（3）匀浆液20000g离心30min。（4）将上清转移到新的离心管中，加入终浓度为10%TCA溶液，4ºC下放置2 h。（5）20000g离心40min，收集沉淀。（6）用冷丙酮洗3次，沉淀在4ºC下干燥，备用。 3. 酚提取法：（1）称取4g果肉，用液氮在研钵中研磨成粉。（2）加入4ml的匀浆缓冲液(匀浆液中含有20 mM Tris-HCl (pH 7.5), 250 mM sucrose, 10 mM EGTA, 1 mM PMSF, 1 mM DTT, 以及1% Triton X-100)，继续研磨。（3）匀浆液20000g离心30min。（4）在上清液中加入等体积的pH7.8 Tris-饱和酚，充分摇荡，混匀，10000g，4ºC下离心30min，上层为水相，中间白色物质为杂质，下层为酚相。（注：当提高匀浆缓冲液中sucrose的浓度到1M时，酚相会出现在上层。）（5）回收酚相，加入5倍体积含0.1M 乙酸铵的预冷甲醇，充分混匀，-20ºC下过夜，沉淀蛋白。（6）沉淀用含0.1M 乙酸铵的预冷甲醇洗2次。预冷丙酮洗2次。（7）沉淀在4ºC下干燥，备用。参考文献： Saravanan R S, Rose J K C. 2004. A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics, 4: 2522-2532. Shen S H, Jing Y X, Kuang T Y. 2003a. Proteomics approach to identify wound-response related proteins from rice leaf sheath. Proteomics, 3: 527-535. Shen S H, Sharma A, Komatsu S. 2003b. Characterization of proteins responsive to Gibberellin in the leaf-sheath of rice Oryza sativa L seedling using proteome analysis. Biol Pharm Bull, 26: 129-136. Chan, Z.L., Qin, G.Z., Xu, X.B., Li, B.Q., and Tian, S.P. 2007. Proteome approach to characterize proteins induced by antagonist yeast and salicylic acid in peach fruit. J. Proteome Res. 6: 1677-1688

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发表于 2012-12-3 14:28 资料个人空间短消息加为好友

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比较完整

双向电泳实验过程及相关溶液配置双向电泳实验过程及相关溶液配置 A．实验过程一、实验原理：2-DE的第一向电泳等电聚焦是基于等电点不同而将蛋白粗步分离，第二向SDS-PAGE是基于蛋白质分子量不同，而将一向分离后的蛋白进一步分离。这样就可以得到蛋白质等电点和分子量的信息。二、实验步骤： 1. 样品的溶解取纯化后的晶体蛋白3.0mg，加入300ul裂解液(1mg蛋白:100ul裂解液)振荡器上振荡10min左右，共处理一个小时。其中每隔10～15分钟振荡一次，然后13200rpm离心15min除杂质，取上清分装，每管70ul，—80oC保存。 2. Bradford法测蛋白含量取0.001g BSA（牛血清白蛋白）用1ml超纯水溶解,测定BSA标准曲线及样品蛋白含量。取7个10ml的离心管，首先在5个离心管中按次序加入0ul, 5ul, 10ul, 15ul, 20ul 的BSA溶解液，另2管中分别加入2 ul的待测样品溶液，再在每管中加入相应体积的双蒸水（总体积为80ul），然后，各管中分别加入4ml的Bradford液（原来配好的Bradford液使用前需再取需要的剂量过滤一遍方能使用），摇匀，2min在595nm下，按由低到高的浓度顺序测定各浓度BSA的OD值，再测样品OD值。(测量过程要在一个小时内完成)。例如：编号蛋白量（ul） Buffer(ul) Bradford(ml) OD595值 1 0 80 4 0 2 5 75 4 0.024 3 10 70 4 0.061 4 15 65 4 0.091 5 20 60 4 0.116 Bt4 2 78 4 0.079 Bt4 4 76 4 转Bt4 2 78 4 0.075 转Bt4 4 76 4 标准曲线方程式：Y= aX+b.其中Y为 OD值，X为蛋白含量。 a、b通过作图输入数据可知相关系数通过输入数据，作图，软件分析可得 OD值测量过程：比色皿用70%的乙醇保存，待用时用双蒸水冲洗，再用无水乙醇冲洗，双蒸水冲洗，再加入待测样品溶液润洗，然后，加入样品，测定OD值。 3. 双向电泳第一向---IEF（双向电泳中一律使用超纯水） 3.1 水化液的制备称取2.0mg 的DTT，用700ul水化液储液溶解后,加入8ul 0.05% 的溴酚兰，3.5ul（0.5%v/v）IPG buffer (pH 3-10)振荡混匀，13200rpm离心15min 除杂质，取上清。在含300ug 蛋白（经验值）的样品溶解液中加入水化液，至终体积为340ul，振荡器上振荡混合,13200rpm离心15min除杂质，取上清。 3.2 点样，上胶分两次吸取样品，每次170ul, 按从正极到负极的顺序加入点样槽两侧，再用镊子拨开Immobiline DryStrip gels (18cm，pH 3—10)胶条，从正极到负极将胶条压入槽中,胶面接触加入的样品。注意：胶条使用前，要在室温中平衡30分钟；加样时，正极要多加样，以防气泡的产生；压胶时不能产生气泡；酸性端对应正极，碱性端对应负极；样品加好后，加同样多的覆盖油(Bio-Rad)，两个上样槽必须与底线齐平。 3.3 IPG聚焦系统跑胶程序的设定（跑胶温度为20oC） S1 (30v, 12hr, 360vhs, step) S2 (500v, 1hr, 500vhs, step) S3 (1000v, 1hr, 1000vhs, step) S4 (8000v, 0.5hr, 2250vhs, Grad) S5 (8000v, 5hr, 40000vhs, step) 共计44110vhs, 19.5小时其中S1用于泡胀水化胶条，S2和S3用于去小离子，S4和S5用于聚焦 3.4 平衡用镊子夹出胶条,超纯水冲洗后，在滤纸上吸干（胶面，即接触样品那一面不能接触滤纸，如果为18cm的胶条要将两头剪去），再以超纯水冲洗，滤纸吸干（再次冲洗过程也可省略），然后用镊子夹住胶条以正极端（即酸性端）向下，负极端（即碱性端）向上，放入用来平衡的试管中（镊子所夹的是碱性端，酸性端留有溴酚兰作为标记），用平衡液A，平衡液B先后平衡15min。注:平衡时要注意保持胶面始终向上,不能接触平衡管壁。平衡第二次时，在沸水中煮Marker 3min,剪两个同样大小的小纸片，长度与一向胶条的宽度等同，然后吸取煮好的Marker，转入SDS—PAGE胶面上，保持紧密贴合；同样在第二次平衡时，煮5%的琼脂糖10ml。 4. 双向电泳第二向---SDS-PAGE 4.1 配胶(两根胶条所用剂量) 分离胶：（T=8% 80 ml）：溶液于真空机中抽气后再加APS和TEMED 30 % 丙烯酰胺储液 21.28ml 分离胶buffer 20ml 10%APS 220ul TEMED 44 ul 双蒸水 38.72ml 浓缩胶：（T=4.8% 10ml） 30 % 丙烯酰胺储液 1.6ml 浓缩胶buffer 2.5ml 10%APS 30ul TEMED 5ul 双蒸水 5.9ml 4.2 灌胶将玻璃板洗净后,室温晾干,然后,将电泳槽平衡好,玻璃板夹好,再在玻璃板底部涂上凡士林以防漏胶,倒入正丁醇压胶,凝胶后(这时会出现三条线),用注射器吸去正丁醇,超纯水洗两次,再用滤纸除水后,倒入浓缩胶,正丁醇压胶,凝胶后,用注射器吸去正丁醇,超纯水洗两次,再加入超纯水,用保险膜封好。 4.3 转移剪两个小的滤纸片，吸取Marker后，放入SDS—PAGE胶面的一端。然后，将平衡好的IPG胶条贴靠在玻璃板上,加少量的5%的琼脂糖溶液在胶面上(琼脂糖凝胶在转移前十几分钟的时候配好,水浴加热溶解,并保持烧杯中水处于沸腾状态,至用之前再拿出来),再将IPG胶条缓缓加入SDS—PAGE胶面,其中不断补加5%的琼脂糖溶液,注意不能产生气泡。 4.4 跑胶浓缩胶 13mA 分离胶 20mA 共约5.5个小时 5. 银染(两根胶条所用剂量)（银染特别注意用超纯水） 5.1 固定 30min 无水乙醇 200ml+乙酸50ml，用超纯水定容至500ml 5.2 敏化 30min 无水乙醇 150ml Na2S2O3•5H2O 1.5688g 无水乙酸钠 34g 先用水溶解Na2S2O3•5H2O和乙酸钠,再加乙醇,最后定容至500ml 5.3 洗涤 5min × 3次 5.4 银染 20min AgNO3 1.25g 用超纯水定容至500ml 5.5 洗涤 1min × 2次 5.6 显影无水Na2CO3 12.5g 用超纯水定容至500ml 甲醛(37%)0.1ml, 临时加 5.7 终止 10min EDTA—Na2•2H2O 7.3g 用超纯水定容至500ml 5.8 洗涤 5min × 3次注：整个双向电泳实验中全部使用超纯水，尽量减少离子的影响。 B．实验相关试剂配制 1． Bradford 工作液 95%乙醇 25ml 先用乙醇溶解考马斯亮兰G250，溶解完后再加磷 85%磷酸 52ml 酸，最后超纯水定容至500ml。过滤后置于棕色瓶考马斯亮兰G250 0.035g 外加油皮纸保存（Bradford不稳定，一周内有效） 2．裂解液尿素 8M 硫脲 2M CHAPS 4% DTT 60 mM Tris—base 40 mM（如果有条件可以添加PMSF 0.5mM和5%的Pharmalate） 3. 水化液储液尿素 8M 硫脲 2M CHAPS 4% Tris—base 40 mM 4. 分离胶buffer (pH8.8) 250ml SDS 0.4% 1g Tris—HCl 1.5M 45.4275g 5. 浓缩胶buffer (pH6.8) 100ml SDS 0.4% 0.4g Tris—HCl 0.5M 6.07g 6.

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发表于 2012-12-3 14:29 资料个人空间短消息加为好友

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凝胶储存液（30%的丙烯酰胺） 250ml Acr 29.2% 73g Bis 0.8% 2g 7. 电极缓冲液（跑一次要配制2500ml）甘氨酸 43.2g 36g Tris 9g 或 7.5g SDS 3g 2.5g 超纯水定容至3000ml 超纯水定容至2500ml 8． 0.5M Tris —HCl pH 6.8储液 6.1g Tris先用30ml超纯水溶解，再用46ml，3M HCl调pH6.8,再加水定容至100ml 9．平衡液储液脲（即尿素） 36g 甘油 30% 30ml SDS 1% 1g 0.5M Tris—HCl pH6.8 10ml 超纯水定容至100ml 10. 平衡液A（一根胶条） DTT 20mg 平衡液储液 10ml 11．平衡液B（一根胶条）碘乙酰氨 300mg 平衡液储液 10ml 0.05%溴酚兰 15ul (平衡液A、B均需临时配制) 12．0.5%琼脂糖10ml 琼脂糖 0.05g 电极缓冲液 10ml 溴酚兰 25ul 补：4、5、6的溶液需过滤后储存于4OC备用。 C．药品 CHAPS 兼性离子去垢剂去垢剂可破坏蛋白质分子之间的疏水相互作用， SDS 离子型去垢剂提高蛋白质的溶解性，防止在等电聚焦时析出尿素离液剂可改变或破坏氢键等次级键的结构，使蛋白质硫脲离液剂变性并使蛋白失活。尿素和硫脲联合使用，可以大大增加蛋白质的溶解性 DTT 还原剂断裂蛋白质分子中Cys残基之间形成的二硫键，增加蛋白质的溶解性。但过分提高DTT的浓度，由于它pKa在8左右，因而会影响pH梯度。DTT在碱性pH下会去质子化，等电聚焦时会损耗，导致二硫键复原，蛋白质沉淀 BSA Bradford中制作标准曲线用无水乙醇和磷酸一起，提供Bradford中的环境磷酸提供Bradford中的酸性环境 Tris 构成缓冲液的成分，可用于抗衡pH的变化 IPG buffer 覆盖液即矿物油，防止水分蒸发，样品干燥。丙烯酰胺（Acr）以丙烯酰胺为单体，甲叉二丙烯酰胺为交联剂，甲叉二丙烯酰胺（Bis）在催化剂（Aps）和引发剂（TEMED）作用下，聚合交联成三维网状结构琼脂糖溴酚兰指示剂作用碘乙酰氨（IAA）平衡液B中使用，中和A液中的DTT 正丁醇比聚丙烯酰胺密度小，用于凝胶制作过程中的压胶甘油无机盐的良好溶剂，热稳定性好 Marker 考马斯亮兰G250（Bradford法用）考马斯亮兰G250有红、蓝两种不同颜色的形式在一定浓度的乙醇及酸性条件下，可配成淡红色的溶液，当与蛋白结合后，产生蓝色化合物，反应迅速而稳定，反应化合物在465~595nm处有最大的光吸收值，化合物颜色深浅与蛋白浓度的高低成正比关系，因此可检测595nm的光吸收值的大小计算蛋白的含量甘氨酸与Tris构成缓冲系统 AgNO3 EDTA 金属螯合剂，可以结合银离子，终止银染过程

xingyi08[使用道具]
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发表于 2012-12-3 14:32 资料个人空间短消息加为好友

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贴东西，顺便让czlong2008修改

2.2 Preparation of total protein extract For protein extraction about 1 g(1 part) of this fine powder was mixed with 5 ml(5 parts) of precipitation solution containing 10% w/v TCA and 0.07% w/v β-mercaptoethanol in cold acetone which was stored at –20°C overnight according to Isaacson et al(2006). Aliquots of suspension were incubated at –20°C for 60 min. After 5, 15, and 30 min the suspension was mixed, and most proteins will precipitate with in 30 min. Precipitated material was collected by centrifugation(18 000 g, 4°C, 15 min), proteins should precipitate as white flakes. After washing twice with acetone containing 0.07% w/v β-mercaptoethanol the precipitates were dried in a cold vacuum centrifuge. The pellets were resuspended using 25 μL lysis buffer(5 M urea, 2 M thiourea, 2% CHAPS, 2% SB3-10, 40 mM Tris, 65 mM DTT and 0.35% carrier amholytes) per milligramme(Wang et al. 2007), extensively vortexed at room temperature, and then centrifuged at 21 000 g for 10 min at 28°C. Protein concentration was determined by the Bradford assay(Ramagli 1999).

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发表于 2012-12-3 14:32 资料个人空间短消息加为好友

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本人一直在使用的蛋白提取方法（适合于植物组织的材料提取），感觉效果很好！呵呵！与大家共享！ 1，取约3g 新鲜植物材料，液氮磨碎，加入30ml左右TCA-丙酮提取液，混合后，放在－20℃一小时。 2，4℃条件下40000g离心力，离心15分钟，取沉淀。 3，从新将沉淀溶解在等体积丙酮-巯基乙醇溶液中，低温-20度放置1小时。 4，从新4℃条件下40000g离心力，离心15分钟，取沉淀。 5，重复步骤（3）-（4）一遍 6，将沉淀真空干燥使得沉淀成粉末状。 7，取干燥后的样品粉末按1mg/30ul溶解液的比例加入蛋白溶解液混合均匀，36度温浴1小时。 8，25度条件下（室温）12000g离心15分钟，吸取上清转移到新离心管中再次离心15分钟取上清。 9，按Bradford法测定蛋白浓度

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发表于 2012-12-3 14:33 资料个人空间短消息加为好友

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前面的朋友提到了bradford法，那我就把我的方法共享一下：简化版bradford蛋白质定量 1，染色液配制：将 G250：0.01%（w/v）、乙醇：4.75%和磷酸：8.5%，用纯净水溶解混合后用滤纸过滤即可，此溶液可以长期保存在室温下。 2，标准蛋白溶液：1mg/ml牛血清蛋（采用0.1mol/L的NaCl溶解牛血清蛋白），-20度下分装在1ml离心管中（最好一次配10ml，然后分装保存，每一次取出一管）。操作流程 1，取7支10ml试管按表1加入相应的溶液。编号 1 2 3 4 5 6 7（样品）牛血清体积（ul） 0 10 20 40 80 100 10 G250 试剂（ml） 3.0 3.0 3.0 3.0 3.0 3.0 3.0 蛋白浓度:ug/ml 0 100 200 400 800 1000 吸光值 2、将以上溶液混合后在595nm下的吸光值，根据吸收值和浓度的关系计算标准曲线。 3、测定待测样品的吸光值，并根据标准曲线计算样品浓度

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发表于 2012-12-3 14:33 资料个人空间短消息加为好友

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2D Gel Electrophoresis Protocol

Lysis Buffer 7M Urea 2M Thiourea 4% CHAPS Add fresh: 20mM Spermine 20mM DTT 1mM PMSF Rehydration Buffer 8M Urea 2% CHAPS Add fresh: 20mM DTT 0.5% IPG Buffer trace amount of bromophenol blue Equilibration Buffer 6M Urea 30% Glycerol 2% SDS 50mM Tris-HCl pH 8.8 EQ Buffer 1 – add fresh 1% DTT EQ Buffer 2 – add fresh 2.5% Iodoacetamide Sample Preparation 1) Remove media from flasks. 2) Add 4mL of trypsin to each flask and place back in incubator for 2-3 minutes. 3) Remove flasks from incubator and add 4mL of media to each flask to inactivate trypsin. 4) Collect cells into 15mL conical tubes and spin @ 3K RPM for 5-10 minutes at RT. 5) Remove supernatant from conical tubes and add 5mL of 1X PBS to each tube. 6) Resuspend cells in 1X PBS in order to remove excess trypsin and media. 7) Spin @ 3K RPM for 5-10 minutes at RT. 8) Remove supernatant from conical tubes and add 1mL of fresh lysis buffer to each tube. 9) Resuspend cells in fresh lysis buffer then place tubes in refrigerator at 4°C for 2-4 hours. 10) Transfer cell lysates into 1.5mL microcentrifuge tubes (Beckman tubes) and spin at 40K RPM for 1 hour at 4°C. 11) Carefully remove supernatant and transfer to clean microcentrifuge tube. 12) Determine protein concentration and store remaining lysate in -80°C freezer. Isoelectric Focusing/The First Dimension 1) Prepare 2.5mL of fresh rehydration buffer for each type of DryStrip. 2) Place samples on ice to thaw and collect 1.5mL microcentrifuge tubes for sample dilution. 3) Remove the necessary number of DryStrips from -20°C freezer. 4) Determine the amount of rehydration buffer necessary for strip length. 5) Add 200-300ug of cell lysate to clean microcentrifuge tubes and add the difference between the total volume and the volume of sample using fresh rehydration buffer. 6) Remove plastic cover from strip holder and set aside. 7) Add rehydration buffer containing sample to the strip holder at the anode (pointed end of the holder). 8) Remove protective cover from DryStrip before placing gel side down into strip holder starting at the anode (pointed end) and laying strip down to the cathode (blunt end). 9) Move strip back and forth in order to spread out rehydration buffer. 10) Make sure that all bubbles are removed from underneath the DryStrip before adding DryStrip Cover Fluid (Mineral Oil). 11) Add cover fluid from one end until it reaches the middle of the strip holder then add from the opposite side so that fluid meets in the middle. 12) Complete the same process for all strips then place the strip holders on IPGphor system. 13) Set up protocol for the length strip that is being used. 14) For 13cm strip: Rehydration at 20°C for 12 hours 50uA per strip 500V for 1 hour 1000V for 1 hour 8000V for 3-5 hours 15) Move onto equilibration step or rinse strip with MilliQ water and place into screw cap tubes for storing in -70°C freezer. Equilibration 1) Prepare 15 mL of fresh equilibration buffers 1 & 2 for each strip. 2) Wash strips with MilliQ water before placing into equilibration buffer 1. If you are removing strip from -70°C, let tube sit on lab bench to thaw strip. When strip is thawed (strip will be clear) place into equilibration buffer 1. 3) Incubate in equilibration buffer 1 for 15 minutes. 4) Remove strip and rinse with MilliQ water before placing into equilibration buffer 2. 5) Incubate in equilibration buffer 2 for 15 minutes. 6) Remove strip and rinse with MilliQ water and place strip on its side on filter paper to allow excess water to drain from strip. Running the Second Dimension 1) Prepare gel by removing water saturated butanol from top of gel and washing with 1X running

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发表于 2012-12-3 14:34 资料个人空间短消息加为好友

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buffer. 2) Cover top of gel with 1X running buffer and lay strip across the top of the gel making sure that the gel is lying flush with the gel and remove any bubbles between the strip and the top of the gel. 3) Remove the running buffer and add warm 1% agarose made with 1X running buffer. 4) Allow the agarose to cool and solidify, which should only take a few minutes, before moving to the electrophoresis apparatus. 5) Add 1X running buffer to the upper and lower buffer chambers and place gel inside apparatus. 6) Run gel for 15 minutes at 10mA per gel then run gel at 25mA per gel for 4-6 hours (until BPB band reaches bottom of gel). 7) Remove gel from plates and stain. 1-D Polyacrylamide Gel Electrophoresis Gel type Sample volume(μl/well) Total well volume(μl/well) Mini Gels 0.75 mm, 10 well 2 to 5 25 Large Gels 0.75 mm, 10 wells 10-140 150 Sample buffer 0.062 M tris-HCl, pH6.8 2% SDS 0.01% Bromophenol Blue 10% Glycerol 5% 2-mercaptoethanol Upper buffer 10% SDS 10 mL 250mM EDTA 4 mL Reservoir buffer 100 mL Nanopure water add enough to 1 L mark Lower buffer Reservoir buffer 100 ml Nanopure water add enough to 1 L mark Store buffers in refrigerator. Sample Preparation 1. Prepare the sample with the sample buffer to a concentration of 1 μg/μl. 2. Heat at 100 °C for 2-3 min in a boiling water bath. Sample loading 1. Remove the comb from the cassette by sliding it slowly with a steady motion straight up. Do not distort or tear any wells. 2. Flush the wells with water to remove residual acrylamide 3. For glass cassettes leave the tape on the side of the gels. 4. Use permanent marker to draw the bottoms of the wells. That will make it easier to load. 5. Insert these plates on the electrophoresis unit. 6. Pour the upper and the lower buffers 7. Load the appropriate amounts of samples. Load sample buffer in any wells that do not contain samples. Gel Visualization Copper Staining 1. Prepare the Copper stain by dissolving 4 g of CuCl2 in 100 mL of water. 2. Wash the gels with nanopure water. 3. Pour the Cu stain on the gel. Make sure that the gel is completely submerged. 4. Rock for 5 minutes. 5. Pour the stain into the waste and wash the gel with water to remove excess copper. 6. Store the gel in water in the refrigerator. Zn Staining 1. Dilute Imidazole (Solution A) 1:10 with water, mix thoroughly. Dilute Zinc Sulfate (Solution B) 1:10 with deionized water, mix thoroughly. Gels will require 100-150 mL so that they are compeletely immersed. 2. Place the gel in a staining container and add Imidazole, solution A. Rock at room temperature for 10 min. 3. Decant Solution A, and add the dilute Zinc sulfate, solution B. Make sure the gel is completely covered to ensure even staining. Rock at room temperature for 30 sec approx while the gel develops. 4. Decant solution B, and add 100 mL of water. Rinse for 3-5 min rocking at room temperature 5. Decant water and replace with fresh water. The gel can be stored like this for days. Silver Staining for Mass Spectrometric Analysis Step Solution per gel Time(min) Fix 25 ml acetic acid, 100ml methanol, 125 ml water 15 Fix 25 ml acetic acid, 100 ml methanol, 125 ml water 15 Sensitization 75 ml methanol, 10 ml sodium thiosulfate(5%), 17 g sodium acetate, 165 water 30 Wash 250 ml water 5 Wash 250 ml water 5 Wash 250 ml water 5 Silver 25 ml silver nitrate(2.5%), 225 water 20 Wash 250 ml water 1 Wash 250 ml water 1 Develop 6.25 g sodium carbonate, 100 ul formaldehyde 250 ml water Stop 3.65 g EDTA 250 water 10 Wash 250 ml Water 5 Wash 250 ml Water 5 Wash 250 ml Water 5 Destaining Silver from the gels WORKING SOLUTION 30 mM Potassium Ferricyanide 99 mg of Potassium Ferricyanide in 10 ml water 100 mM Sodium Thiosulfate 248 mg Sodium Thiosulfate in 10 ml water Mix them together. Make fresh for use. Add 30-50uL working solution to cover the gel bands and vortex

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发表于 2012-12-3 14:35 资料个人空间短消息加为好友

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occasionally. Repeat till there is no more brown color left. Rinse with water , twice. Cover the gel with 200mM ammonium bicarbonate for 20 min. Discard the liquid to waste. Wash with ACN till the gel pieces are opaque. Dry the pieces under vacuum for 30 min Alternative Version: Silver Staining for Mass Spectrometric Analysis (Use 5 gel volumes of each reagent for staining) Step Solution per gel Time(min) Fix 5% acetic acid; 45% methanol; 50% water 90 Wash water 10 Wash water 10 Sensitization 0.02% sodium thiosulfate 3 Wash water 0.5 Wash water 0.5 Silver 0.1% silver nitrate 30 Wash water 0.5 Develop 0.02% formaldehyde/2.5% sodium carbonate 2 (bands suitable for MS analysis will appear within 30-60 seconds) Stop 1% acetic acid 10 Wash water 20 Wash water 20 Alternate Version: Destaining Silver from the gels WORKING SOLUTION 30 mM Potassium Ferricyanide 99 mg of Potassium Ferricyanide in 10 ml water 100 mM Sodium Thiosulfate 248 mg Sodium Thiosulfate in 10 ml water Mix at 1:1 ratio. Make fresh for use. Cover the band in working solution and vortex until brown color removed (5 minutes) Remove the solution and wash three times with 200 μL water. In-gel Digestion 100 mM Ammonium Bicarbonate 0.79 g Ammonium Bicarbonate Make up to 100 mL with water 10 mM DTT 15.4 mg Dithiothreitol Make up to 10 mL with 100 mM ammonium bicarbonate 55 mM Iodoacetamide 102 mg Iodoacetamide Make up to 10 ml with 100 mM ammonium bicarbonate 0.1 μg/μl Trypsin 200 μl of 25 mM Ammonium Bicarbonate 20 μg trypsin Excise gel bands prior to in-gel digestion

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