http://www1.gelifesciences.com/APTRIX/upp00919.nsf/content/23E7268B06D81881C1256EB400483DC8?OpenDocument&Path=Catalog&Hometitle=Catalog&entry=4&newrel&LinkParent=C1256FC4003AED40-4FE9D73C05DE40CFC12570D90042178C_RelatedLinksNew
The exclusion limit is the molecular weight of the smallest molecule which cannot enter the pores of the matrix. All molecules bigger than the exclusion limit elute in the void volume. The exclusion limit means that the sample is no longer fractionated and will be excluded from entering the pores. This limit will be just above the upper limit of the fractionating range.
The exclusion limit is an extrapolated value defined by convention.
2009年5月15日星期五
2009年4月29日星期三
Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell Instruction Manual(western)
Note
To insure the best performance from the Trans-Blot SD semi-dry electrophoretic transfercell, become fully acquainted with these operating instructions before using the cell to transfer samples. Bio-Rad recommends that you first read these instructions carefully. Then assemble and disassemble the cell completely without transferring sample. After these preliminary steps, you should be ready to transfer a sample.
Bio-Rad also recommends that all Trans-Blot SD cell components and accessories be cleaned with a suitable laboratory cleaner (such as Bio-Rad Cleaning Concentrate, catalog number 161-0722) and rinsed thoroughly with distilled water, before use.
Warranty
Bio-Rad Laboratories warrants the Trans-Blot SD semi-dry electrophoretic transfer cell
against defects in materials and workmanship for 1 year. If any defects occur in the instrument during this warranty period, Bio-Rad Laboratories will repair or replace the defective parts free.The following defects, however, are specifically excluded:
1. Defects caused by improper operation.
2. Repair or modification done by anyone other than Bio-Rad Laboratories or an authorized agent.
3. Use of fittings or other spare parts supplied by anyone other than Bio-Rad Laboratories.
4. Damage caused by accident or misuse.
5. Damage caused by disaster.
6. Corrosion due to use of improper solvent or sample.
This warranty does not apply to parts listed below:
1. Platinum plate electrode.
For any inquiry or request for repair service, contact Bio-Rad Laboratories after confirming the model and serial number of your instrument.
Section 1
Introduction
Blotting was first performed by Southern in 1975 with the transfer of DNA from agarose gels to nitrocellulose membranes. Blotting has subsequently been applied to RNA and protein from both agarose and polyacrylamide gels. Membrane materials have been expanded to include PVDF for improved protein binding capacity. To overcome the inefficiency of capillary transfers, electric current has been adopted for eluting proteins from polyacrylamide gels, as first described by Towbin et al. in 1979. Since that time, electrophoretic transfer has also been used for DNA and RNA blotting.
For blotting PCR fragments, plasmid and vector DNA, and RNA with the SD cell, use theTrans-Blot SD DNA blotting kit. DNA or RNA can be blotted from agarose gel to Zeta-Probe GT membrane in only 10 minutes, without any gel pretreatments. The kit comes complete with DNA/RNA blotting accessories and a detailed instruction manual.
Semi-dry blotting was first reported by Kyhse-Andersen in 1984. Blotting was performed with plate electrodes in a horizontal configuration. The gel and nitrocellulose membrane were sandwiched between sheets of buffer-soaked filter paper, which served as the ion reservoir and replaced the buffer tank. The plate electrodes, separated only by the filter paper stack, provided high field strength (V/cm) across the gel, and very efficient, rapid transfers.
The Trans-Blot semi-dry transfer cell incorporates the original concepts of semi-dry blotting along with innovative features for quick set-up and ease of use. The platinum-coated titanium and stainless steel electrode pair provides efficient, background-free blotting with trouble-free service.
Section 3
Safety Instructions
Read the entire manual before beginning electrophoretic transfers.
Electrophoretic transfer of proteins and nucleic acids is dependent on many factors. Observe the following guidelines to avoid mishaps that may result in serious damage to the instrument or injury to the operator.
1. Do not reverse polarity on this instrument.
This will result in corrosion and rusting of the stainless steel cathode. If this should occur, the stainless steel should be cleaned with a mild abrasive cleaner to remove the rust.
2. Do not exceed 25 V with this instrument.
This could damage the electrodes.
3. Do not adjust the pH of transfer buffers unless specifically indicated.
Follow instructions carefully. Adjustment of pH of transfer buffers, when not indicated, will result in increased buffer conductivity. This is manifested by a higher than expected initial current output as shown by the power supply's current meter. Monitor buffer resistance with the Model 200/2.0 power supply prior to each run to insure proper buffer conductivity.
4. Lengthy transfer times are not recommended.
Do not leave this instrument unattended. Joule heat can be generated rapidly during semi-dry blotting. Transferring longer than 2 hours can damage the unit.
5. Power supply requirements.
The Trans-Blot SD cell should only be used with the microprocessor-controlled Model 200/2.0 power supply (catalog numbers 165-4761 and165-4762), or the Model 1000/500 power supply (catalog numbers 165-4710 and 165-4711). Do not use the Model 250/2.5 power supply with this apparatus. The low voltage, high current operating conditions of the Trans-Blot SD cell are not compatible with the Model 250/2.5 power supply, and will cause the power supply to blow a fuse.
6. Do not operate this instrument in ambient temperatures exceeding 50 °C.
Important
This Bio-Rad instrument is designed and certified to meet IEC 1010-1* safety standards. Certified products are safe to use when operated in accordance with the instructtion manual. This instrument should not be modified in any way. Alteration of this instrument will:
• Void the manufacturer's warranty
• Void the IEC1010-1 safety certification
• Create a potential safety hazard
Bio-Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than for which it is intended or by modifications of the instrument not performed by Bio-Rad or an authorized agent.
*IEC 1010-1 is an internationally accepted electical safety standard for laboratory instruments.
To insure the best performance from the Trans-Blot SD semi-dry electrophoretic transfercell, become fully acquainted with these operating instructions before using the cell to transfer samples. Bio-Rad recommends that you first read these instructions carefully. Then assemble and disassemble the cell completely without transferring sample. After these preliminary steps, you should be ready to transfer a sample.
Bio-Rad also recommends that all Trans-Blot SD cell components and accessories be cleaned with a suitable laboratory cleaner (such as Bio-Rad Cleaning Concentrate, catalog number 161-0722) and rinsed thoroughly with distilled water, before use.
Warranty
Bio-Rad Laboratories warrants the Trans-Blot SD semi-dry electrophoretic transfer cell
against defects in materials and workmanship for 1 year. If any defects occur in the instrument during this warranty period, Bio-Rad Laboratories will repair or replace the defective parts free.The following defects, however, are specifically excluded:
1. Defects caused by improper operation.
2. Repair or modification done by anyone other than Bio-Rad Laboratories or an authorized agent.
3. Use of fittings or other spare parts supplied by anyone other than Bio-Rad Laboratories.
4. Damage caused by accident or misuse.
5. Damage caused by disaster.
6. Corrosion due to use of improper solvent or sample.
This warranty does not apply to parts listed below:
1. Platinum plate electrode.
For any inquiry or request for repair service, contact Bio-Rad Laboratories after confirming the model and serial number of your instrument.
Section 1
Introduction
Blotting was first performed by Southern in 1975 with the transfer of DNA from agarose gels to nitrocellulose membranes. Blotting has subsequently been applied to RNA and protein from both agarose and polyacrylamide gels. Membrane materials have been expanded to include PVDF for improved protein binding capacity. To overcome the inefficiency of capillary transfers, electric current has been adopted for eluting proteins from polyacrylamide gels, as first described by Towbin et al. in 1979. Since that time, electrophoretic transfer has also been used for DNA and RNA blotting.
For blotting PCR fragments, plasmid and vector DNA, and RNA with the SD cell, use theTrans-Blot SD DNA blotting kit. DNA or RNA can be blotted from agarose gel to Zeta-Probe GT membrane in only 10 minutes, without any gel pretreatments. The kit comes complete with DNA/RNA blotting accessories and a detailed instruction manual.
Semi-dry blotting was first reported by Kyhse-Andersen in 1984. Blotting was performed with plate electrodes in a horizontal configuration. The gel and nitrocellulose membrane were sandwiched between sheets of buffer-soaked filter paper, which served as the ion reservoir and replaced the buffer tank. The plate electrodes, separated only by the filter paper stack, provided high field strength (V/cm) across the gel, and very efficient, rapid transfers.
The Trans-Blot semi-dry transfer cell incorporates the original concepts of semi-dry blotting along with innovative features for quick set-up and ease of use. The platinum-coated titanium and stainless steel electrode pair provides efficient, background-free blotting with trouble-free service.
Section 3
Safety Instructions
Read the entire manual before beginning electrophoretic transfers.
Electrophoretic transfer of proteins and nucleic acids is dependent on many factors. Observe the following guidelines to avoid mishaps that may result in serious damage to the instrument or injury to the operator.
1. Do not reverse polarity on this instrument.
This will result in corrosion and rusting of the stainless steel cathode. If this should occur, the stainless steel should be cleaned with a mild abrasive cleaner to remove the rust.
2. Do not exceed 25 V with this instrument.
This could damage the electrodes.
3. Do not adjust the pH of transfer buffers unless specifically indicated.
Follow instructions carefully. Adjustment of pH of transfer buffers, when not indicated, will result in increased buffer conductivity. This is manifested by a higher than expected initial current output as shown by the power supply's current meter. Monitor buffer resistance with the Model 200/2.0 power supply prior to each run to insure proper buffer conductivity.
4. Lengthy transfer times are not recommended.
Do not leave this instrument unattended. Joule heat can be generated rapidly during semi-dry blotting. Transferring longer than 2 hours can damage the unit.
5. Power supply requirements.
The Trans-Blot SD cell should only be used with the microprocessor-controlled Model 200/2.0 power supply (catalog numbers 165-4761 and165-4762), or the Model 1000/500 power supply (catalog numbers 165-4710 and 165-4711). Do not use the Model 250/2.5 power supply with this apparatus. The low voltage, high current operating conditions of the Trans-Blot SD cell are not compatible with the Model 250/2.5 power supply, and will cause the power supply to blow a fuse.
6. Do not operate this instrument in ambient temperatures exceeding 50 °C.
Important
This Bio-Rad instrument is designed and certified to meet IEC 1010-1* safety standards. Certified products are safe to use when operated in accordance with the instructtion manual. This instrument should not be modified in any way. Alteration of this instrument will:
• Void the manufacturer's warranty
• Void the IEC1010-1 safety certification
• Create a potential safety hazard
Bio-Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than for which it is intended or by modifications of the instrument not performed by Bio-Rad or an authorized agent.
*IEC 1010-1 is an internationally accepted electical safety standard for laboratory instruments.
北京A类定点医疗机构（2009）
1、首都医科大学附属北京同仁医院；
2、首都医科大学宣武医院；
3、首都医科大学附属北京友谊医院；
4、首都医科大学附属北京中医医院；
5、首都医科大学附属北京朝阳医院；
6、首都医科大学附属北京天坛医院；
7、北京大学第一医院；
8、中国医学科学院北京协和医院；
9、北京大学人民医院；
10、北京大学第三医院；
11、北京积水潭医院；
12、中国中医研究院广安门医院；
13、中日友好医院；
14、北京大学首钢医院；
15、北京市健宫医院；
16、北京市房山区良乡医院；
17、北京市大兴区人民医院；
18、北京市石景山医院。
19、北京世纪坛医院（北京铁路总医院）
2、首都医科大学宣武医院；
3、首都医科大学附属北京友谊医院；
4、首都医科大学附属北京中医医院；
5、首都医科大学附属北京朝阳医院；
6、首都医科大学附属北京天坛医院；
7、北京大学第一医院；
8、中国医学科学院北京协和医院；
9、北京大学人民医院；
10、北京大学第三医院；
11、北京积水潭医院；
12、中国中医研究院广安门医院；
13、中日友好医院；
14、北京大学首钢医院；
15、北京市健宫医院；
16、北京市房山区良乡医院；
17、北京市大兴区人民医院；
18、北京市石景山医院。
19、北京世纪坛医院（北京铁路总医院）
2009年4月26日星期日
2009年4月25日星期六
2009年4月23日星期四
2009年4月21日星期二
我早告诉你了，上最后一节课的是韩国人
耶路撒冷寒风习习，这是地球上有史以来最悲惨的一天，就在这一天，耶稣基督告诉他的门徒，他将要去赴死。以利的心情是又痛苦又欣喜。 他不是耶稣的十二门徒，他要高于十二门徒——他和耶稣从小长到大，简直就是他的死党，也只有他才真正地知道，耶稣的老爸就是上帝。 “夫子，离开耶路撒冷，我们去伯利恒吧，我们不能失去您！”一次聚餐时，以利曾这样恳求他。 “不行。”救世主回答说，“我的命运已经被注定，我的死期近了。” “为什么？” “为了拯救世人。” “但是您活着可以救更多的人！” “闭嘴！”耶稣恼怒地说，“我从上头下来时，这件事情就定下了，任何人无法更改。我的下放实习期只有那么长时间。” 以利花了很长时间来接受这个现实，最后他小声地提了一个问题：“您会怎么离开我们？” 救世主耶稣基督挠挠头：“这个事情么……老爸倒没明文规定。他希望我来个轰轰烈烈的死法把事情闹大一点，比如坐在马车里被人刺杀搞起 一场战争什么的。但难得死一次，我可不想死得那么难受。” “我的主，您想怎样行事？”以利急切地问。 “恩……或许上个法庭，在舒适的牢房里住上两个星期，最后在与朋友们谈论哲学的时候喝下毒酒是个比较好的选择。”救世主开心地说，“ 很有文化气息，更重要的是，不疼。把那杯麦酒拿过来，再撕一块面包给我。” ———————————————————————————————————————酒过三巡，耶稣拿着几枚铜币偷偷离席了。别人都不知道他的去向，只有身为耶稣死党的以利知道，他大概又去找那个叫玛利亚，来自抹大拉的妓女了。门徒们见耶稣离开了，于是也纷纷散去。以利独自走在乡间的小路上，一路上怅然所失，直到一个声音在他耳边响起。 “以利！”那个声音苍老而慈祥。 “谁？”以利转身四顾，小路上一个人都没有。 “我从上头来的。”那个声音简单地说道。 以利敬畏地跪下去，脑袋中的绝大部分都被虔敬所充满，剩下的那块脑细胞则在暗自欣喜——自己终于当先知了。 “我很忙，”上头的声音说，“咱们长话短说，你是我儿子的朋友是吗？” “是的，主。” “他今天刚刚告诉你，他的下放实习期就快结束了？” “是的，主。” “他告诉你他打算怎么回来吗？” “是的，主。夫子他打算经受一场牢狱，最后边谈论哲学边喝毒酒。” “我早告诉他过，这种死法已经有人用过了。”上头的声音似乎有点小生气，“他还是不肯用我建议的方法吗？” “马车里那个方法？他不肯，主。” “也罢，这个点子我很喜欢，迟早我要来一次。”上头的声音突然严肃起来，“你要告诉他，他的实习成绩很差。他的绩效指标是作王一千年，但现在连他家乡的人都不听他的话，如果不能靠这次的死法赚回一点分数，那他的成绩就是不及格。” 以利敬畏地回答，“我明白了，主。” “不，你还不明白。我要你帮忙去说服他换个流芳百世的死法。” “主，夫子怎么可能听我的呢？您为什么不直接和他说呢？” “白痴！”上头的声音怒了，“哪个青春期的儿子肯听老爸的话？比起老爸来，兄弟的话更有说服力！” “那么，请主赐给我伶俐的舌头好去说服夫子。”以利乞求道。 “你要的不是舌头，而是知识……”上头的声音似乎在考虑什么，“我决定了，我把你送去上学，回来后你就能替他设计出一个完美的死法了。” “主……可是……” “我都替你操办好了，去吧！”上头的声音刚落，以利就觉得一道光笼罩住了自己，大地、重力及整个世界都消失了。在晕厥过去之前，以利听到的最后一句话是，“替他赢过默罕默德那小子！” ———————————————————————————————————————正如同以利不知道自己什么时候开始会说美式英语一样，尤斯佛教授也不知道自己班里什么时候进了一个巴勒斯坦插班生。前两天那个穆斯林插班生来的事情她也完全事先不知情，她有点恼火，看来什么时候要找教务处的人好好谈谈。她检查了一下这个新学生的资料，所有的证件、证明一应俱全，而且看落款，全部都是同一天内签发出来的，她耸耸肩，官僚机构几个世纪来从未如此高效率过，看来这个插班生上头有人。 “以利同学，欢迎进入新奥尔良商学院，我是尤斯佛·摩马斯特，”尤斯佛教授欢迎他说，“不知道你之前在哪所学校就读？” 以利还在思索旧奥尔良是个什么东西，突然被问道之前的学校，他只能支吾地说：“恩……恩……我原来在伯利恒那边……” “以色列的圣玛门商学院？”尤斯佛教授设法掩饰她的鄙夷之情，“他们的东西太理论化，我这边才是真正实用的东西。” “恩……实用？”以利想到了他的任务，“尤斯佛教授，你们这边还研究怎么弄死人吗？” 尤斯佛教授眨了眨眼睛，“弄死人？”她想到了当下的次贷危机，以及持续上扬的失业率和自杀率，“对，学商的人都很擅长。以利，我喜欢你的幽默。” “真的？”以利兴奋了，“那赶紧上课吧，我赶时间。” ———————————————————————————————————————新奥尔良商学院一向标榜自己“网络全球人才”，它的师资力量来自世界各地，以利上的第一堂品牌运营课的老师崔迪逊先生就是一个英国人。“品牌，”崔迪逊先生在课堂上说，“就是对旧有传统的实体化。旧有的传统是虚无空泛的，只有把它凝聚成品牌，才能具有顽强的竞争力。”他指着教室角落里的一个穆斯林打扮的学生，“穆萨先生，给我们据个例子，说明下新生品牌是如何打败其他旧有传统的？” 这个叫穆萨的学生似乎有点心不在焉，“恩……恩？”他说。 崔迪逊先生脸色有点不好看，他对穆萨的同桌说：“你来替他说？” “恩……旧有传统？”这个同桌想了下，“有名目的宗教取代了无名目的先祖信仰？” “虽然不是个商业上的例子，但还是很贴切。”崔迪逊先生脸色好看了点，“以信仰为例，当宗旨明确的宗教出现后，部落的先祖信仰就消逝了，因为前者有名字，有品牌，而后者什么都没有。” “您能具体说说吗？”以利发言道，“有关宗教，比如耶稣的宗教？” “注意你的话，以利先生。”崔迪逊先生正视他道，“搞商业的不能牵扯到任何信仰与道德，我们在这里不能谈论这些，你刚才说的那个名字也不能再提。” 他的死亡必须成为品牌。以利在笔记本上记下了这句话，用的是漂亮的古希伯来花体字。———————————————————————————————————————“你刚才到哪里去了？”耶稣生气地说，“这个月是你负责的采购，但我们的酒都喝完了！” “恩……我主，您不是能把水变成酒吗？”以利怯生生地回答。 “我只能变葡萄酒，”救世主气鼓鼓地回答，“但我更喜欢大口地喝麦酒！你刚才去哪里了？一整天都见不到你人。” 以利回答说：“我蒙召去替上帝办差了。” “老爸？他要你干什么？”耶稣的语气充满疑惑。 “恩……我被主送到一个神奇的地方，上了几节课。一下课，我就又被送回来了。” “上什么课？” “恕我直言，”以利知道不应该欺骗救世主，于是就壮着胆子直说了，“那里的教授教我怎么安排您的死亡。” “教授？”耶稣露出茫然的神色。 “相当于我们这边的拉比。” 耶稣恍然大悟：“拉比们教了你什么？” “恩……”他努力回想着，“您的死亡必须成为品牌。” “品牌是什么意思？”耶稣问。 “额……”以利的脑门渗出了一层细汗，说实话，几个小时的课上，崔迪逊先生根本没解释什么叫“品牌”——在他看来，这个太基本了，不值得浪费口舌再解释一遍。“品牌……大概就是招牌。” “招牌？挂在酒馆门口的那种？” “对，我主，这样的话，所有的人都会看到您的牺牲，他们都会被感动，然后汇聚到您的名下。” “好主意！”耶稣赞许道，“我要设计一个与众不同的招牌，任何人看到后，都能铭记我做出的牺牲，直至千年。” “如您所愿。”以利深深下拜，然后赶回了自己的家。他要早点睡觉，明天一早还要去上课呢。 ———————————————————————————————————————“别听昨天那约翰牛的废话。”经营策略课是由尤斯佛教授自己来讲的，她似乎对崔迪逊先生上节课说的品牌传统论相当不屑。“人如其名，崔迪逊先生非常非常地传统——换一句话说，他已经落伍了。想获得成功的人，都必须学会蔑视传统。” “为什么这么说？”下面有人提问。 “很简单，每个人都有自己叛逆的欲望。”她回答道，“但限制于传统的束缚，很少有人敢于充分表现自己的叛逆面。但如果有谁做了别人都想做而不敢做的事情，那么他就会成为公众偶像。佐罗本质上只是个强盗，但他却成了所有人的偶像。” “您说的是一个虚构的人物。”有学生反对道。 “当然，但不可否认，监狱里的男性囚犯被女性求爱的概率是自由男性的四倍。”尤斯佛教授说，“这是有权威调查数据支持的，因为大家都觉得一个叛逆的男人更具有吸引力，特别是性吸引力。” “您能解释一下为什么违反传统会产生吸引力吗？”以利举手问道。 “很容易解释。我还准备了道具。以利先生，请站过来，面对着大家，好的，谢谢。”她引导以利站到讲台前面，以便让大家能看到他的脸部表情。“给你看几张照片。” 她出示了几张照片，其中有特雷莎修女、南丁格尔、居里夫人、维多利亚女王，都是一本正经、裹得严严实实的老女人。以利疑惑不解地浏览着这些图片，纳闷地揣测着尤斯佛教授的意图。 突然，一个年轻女人的照片出现在眼前——她嘴唇上一颗明显的美人痣、扎了一个雪白色的马尾辫，胸前有两个巨大的圆锥形物体向前突出，摆出了一个撩人的姿势。以以利的观点来看，衣着相当暴露。他的眼睛瞪得大大的，嘴巴不由自主地张了开来，有透明的液体自嘴角低下。以利保持这这个样子一动不动，半响说不出话，思维已经完全被这个女人带来的视觉冲击所击垮了。他甚至没有注意到台下的爆发的哄笑声。 “以利先生的反应……哈哈哈……比我想象的强烈得多，他一定……哈哈哈……是个老实人……” 尤斯佛教授一边徒劳地克制着爆笑的冲动，一边挣扎地把这句话说完：“麦当娜就是打破传统而成功的一个例子，大家明白了吗？哈哈哈哈……” 台下根本没人听她的，所有人都笑成一团，除了以利以外。后者正呆呆地看着麦当娜的照片，眼睛和嘴巴都张得大大的，一动不动。 更正一下，所有人都笑成一团，除了以利和穆萨以外。后者的表情和以利一摸一样，只不过他坐在教室最后排，没有人注意到他。 ———————————————————————————————————————当以利回到耶稣住处的时候，他着实被吓了一跳。好多人围在耶稣家门口，大声嚷嚷着什么。 “叫老板出来！我们要买酒！”一个醉醺醺的醉汉大声叫嚷着。 耶稣叉着腰堵在家门口，很生气地对围观地人说道：“再和你们说一遍，这里没老板！我们不是卖酒的！” “如果不是卖酒的，为什么要挂这种招牌出来？”另一个醉汉指着耶稣家门口挂着的一块牌子，生气地说道。 耶稣很窘迫的样子，脸涨得通红，“反正我们什么都不卖！你们到别家去买酒吧！” 以利挤到近处一看，他家门口不知什么时候果然多出一块木头招牌，上面画着一个金黄色的酒杯，周围还描绘上了光芒。 耶稣看见以利，示意后者赶紧来帮自己一把。以利义不容辞地挤到耶稣身边，花费了许多口舌，好不容易把众人劝散了，以利赶紧把招牌给取下来了，以免惹出更大的麻烦。“我主，您怎么会把这么一个招牌放在外面的？”他问。 耶稣的窘态更明显了，他支支吾吾地回答：“你不是告诉过我，我的死亡必须成为招牌吗？我就自己做了一个……” 一滴冷汗从以利的额头滑下：“这杯子是？” “额……是毒酒的象征。”耶稣正色道，“这群愚民竟然认为我是卖酒的！” “主啊，这的确与街口酒店的招牌很神似。” “哪里神似啦？比如说……恩……恩，”耶稣上下打量着招牌，“他们的酒杯可没有金黄色的光芒！” 以利决定不在这个问题上继续纠缠下去。“请容我说，主啊，您还决定采用那个死法吗？”他说道，“我觉得那不适合您。” “没有人能够让人子改变决定。”耶稣断然地说，“但是，我主，我有了一个更好的方法。” “虽然我不会听，但你还是说吧。” “您觉得十字架如何？”以利充满期望地说，“风格叛逆，十字架容易做成招牌，而且围观者众多。” 耶稣皱皱眉头，“那是强盗的死法。” “您说对了！”以利回答，“那有助于增加您的阳刚之气，并且可以吸引舆论的注意。”“会被脱光的！”耶稣抗议道。 “那会吸引很多女信徒。”以利说。 “很疼！”耶稣抗议道。 “我可以调点麻药给您喝。”以利说。 “在十字架上会上一两天都死不掉！”耶稣抗议道。 “我有个士兵朋友，他可以扎您一枪。”以利说。 “只有强盗才能上十字架！”耶稣抗议道。 “我可以帮您安排，我认识罗马少尉朗基努斯。”以利说。 “……”耶稣无声地抗议道。 以利毫不畏缩地盯着他看。 “反正我就要喝毒酒死，谁也别管我。”耶稣被盯得受不了了，他气鼓鼓地站起来，也不管以利，径自出门右转走掉了，大概又是去找抹大拉的玛利亚去了。 当以利独自在房间里不知所措的时候，一个熟悉的声音从上头降临到他的脑海“看来你也说服不了他嘛。” “我让您失望了，主。求您宽恕我。”以利跪下祈祷。 “相反，我对你的想法很有兴趣。”上头说，“我把我的独子交到你手里了，你可以任意待他。” 圣灵从天上降下落到以利身上，以利觉得自己被一种邪恶的勇气所充满了——他要把救世主钉上十字架。 ———————————————————————————————————————沟通交流课的老师据说来自中国淳朴的农村，但自从在一个叫“墙壁街”的地方浸淫数十年以后，郝先生就完全是一副“墙壁街”特有的样子了。 “人们常说，沟通反映现实，但俺却不这么认为。”郝先生的英语仍然带有他故乡的乡音，“在俺的观念里，沟通创造现实！” “什么叫沟通创造现实呢？”郝先生解释道，“俺们可以举哈根达斯的例子。你们谁没吃过哈根达斯冰激淋的，举手！” 以利左右看看，没有人举手，于是他就压抑下了举手的冲动。但是坐在后排的穆斯林穆萨先生举手了。 “好小伙，喏，接着！”郝先生用大拇指弹了一个一美元硬币给穆萨，“出门右转的路边小店，自己去买一桶尝尝吧。大家都知道，哈根达斯在这里是一种极其低档而普遍的品牌，但到中国去卖的价格，甚至超过了美国本土。但是那样离谱的高价，都有无数中国人去买，大家知道为 什么吗？请你回答，穆萨先生？” “呃……呃……”穆萨依然是一副茫然的表情。 郝先生摇摇头，用只有前排才听得到的声音嘟哝道：“肯定是乡下来的。” 然后他换上笑容，大声说：“不知道也没关系，答案很简单，它暗示中国人，哈根达斯是世界著名的贵族品牌。也就是说，它本身是什么无所谓，真正有所谓的是它告诉别人它自己是什么。” 以利发言说，“我告诉别人的任何事情，别人都会相信？这不太可能吧。” “当然不会，我们还要学会伪装，也就是说至少要看上去可信。” “如果这件事情很难伪装呢？” “ 那就把水搅浑。”郝先生愉快地说，“股票市场有那么多满口术语的金融分析师，你以为他们靠什么赚钱？他们的意见都是一半对一半的，如果真要追究预测成功率，至少有一半的分析师要下岗。他们的策略就是，用混乱的术语与复杂的计算关系把原本简单的股市搅浑，这样别人就搞不清楚他们谁的意见更正确，然后他们就都能持久地从中牟利了，这是一个集体骗局。” “骗局？这不道德吧。”以利皱皱眉头。 “道德？俺们搞经济的要啥道德！”郝先生用这句斩钉截铁的话结束了对话。 ———————————————————————————————————————那天放学回到耶路撒冷后，以利又和上帝聊了一次。借助上帝时间旅行的能力，以利在一个同傍晚之内做了很多安排。因为这些事情是同时发生的，所以一一记录如下： 一、“为了使基督更有慷慨赴死的哲学家气质，”以利给耶稣上了堂“行为礼仪课”。比如告诉耶稣，哲学家通常都喝葡萄酒而不喝卖酒，通常都管葡萄酒为自己的血，面饼则是自己的身体等等。当然，哲学家的这些特点都是以利虚构的。 二、他找到了耶稣十二弟子之一的犹大，向他透露了耶稣与抹大拉的玛利亚的长期暧昧关系。暗恋玛利亚的犹大悲愤异常，誓与耶稣老匹夫势不两立。以利又问犹大，为玛利亚赎身筹的钱还差多少，犹大回答还差三十银币。 三、他化妆去了趟大祭司的家，绘声绘色地描绘了耶稣在自家门口鼓动暴民、企图破坏宗教秩序的异端行为。大祭司向自己手下一打听，果有此事，而且参与的暴民各个都是脾气暴烈的酒鬼，一旦造起反来危害甚大。大祭司感谢以利主动履行好市民的责任，请他作为内应，拘捕耶稣。以利婉拒，并提议说耶稣犯罪集团中的骨干成员犹大可以收买，作价三十银币。 四、以利去拜访了当时在耶稣门口吵闹的那群酒吧顾客，诓称耶稣因为卖甲醇酒毒死人被抓了，不日即将受刑。顾客们表示一旦时间地点确定，便会来旁观。 五、以利再次拜访了耶稣，告诉他说，他和玛利亚的秘密暧昧关系被犹大发现了，后者正把这内幕透露给吟游诗人协会，以换取稿费。耶稣大骂犹大不地道，出卖师傅。 六、耶稣接受了以利的建议，决定召开一个派对，邀请十二位弟子赴宴。他计划在宴会上喝毒酒告别人世，同时拜托以利写本《以利篇》的对话集。以利替耶稣跑腿发了请帖，但却把写书的重担扔给了大徒弟西门保罗，而保罗也懒得写，把这事情推给了马可、马太、路加、约翰这四个小弟。 七、由于以利的幕后撮合，犹大和大祭司的管家进行了良好而秘密的磋商，双方在友好的气氛下就出卖耶稣等一系列问题达成了共识。 八、以利最后去拜访的，是他小时候的邻居，罗马少尉朗基努斯，后者爽朗地答应了朋友的请求。 ———————————————————————————————————————一切都结束后，以利就停了一切的工，躺下休息。他躺在自己的床上，悠闲地想，骨牌都已经摆放到位，只需要轻轻推一下，一切就会按计划进行。一切都进行得太完美了。 他正想着，上头的声音又突然响起：“以利，你已经安排好了吗？” 以利翻身拜倒：“是的，主。” “新奥尔良商学院的课程还没结束，明天你不回去上课了吗？” “不需要了，主。”以利回答，“我的任务已经完成，一切都安排好了。” “明天上课的可是韩国人哦~” “不需要了，主，明天我要目睹我主升天。” 上头叹了口气，“好吧，但愿一切顺利。” ———————————————————————————————————————如祂所愿，一切出奇地顺利。耶稣的告别派对从晚餐开始，一直High到通宵达旦。“这是我的血，你们拿去喝。”醉熏熏的耶稣把葡萄酒递给门人，“这是我的身体，你们拿去吃。”他边说，边撕下一片面包，借着酒劲扔向犹大。 犹大正郁闷地坐在角落里，心里还犹豫是不是要背叛耶稣，猛不丁地被面包砸中了脸，顿时就火了，站起来想看是谁扔的，然后他就看见了耶稣醉红的脸正冲着自己不坏好意地微笑。犹大强忍怒火：“您喝醉了，夫子。” 耶稣看见犹大的脸，脑子里想象着犹大靠出卖基督的绯闻而换取金钱的样子，不觉心中愠怒。“你们中有人卖我了！”他突然大声喊道。 门徒们非常惊慌，一个个地问，“夫子，是我吗？” 犹大也发问道：“夫子，是我吗？” 耶稣直视着他的眼睛，“你说得是。” 犹大心中一慌，既然基督已经知道了，犹大就再也没有犹豫的余地了。他悄悄从派对中离开，回来时，带领着大祭司及罗马士兵。 ———————————————————————————————————————经过一夜审讯，第二天一早，耶稣就被判暴乱罪，钉上十字架处死。“我不能喝毒酒而死吗？”耶稣委屈地说，“我费心营造了一晚上的气氛，你们再晚一会冲进来抓人，我就已经把毒酒喝掉了。” “想自杀来逃避审判，哪那么容易！”负责抓捕的朗基努斯少尉回答道，“来人，把他的十字架给他，他要自己背上山！” 有人把十字架抬过来了，耶稣一看，就几乎昏倒——那十字架用最好的橡木制作的，做工精良气势雄伟，比一般的十字架更高大更结实，重量更是达到了一般十字架的四五倍。 “军爷，”耶稣带着哭腔问，“能给我个普通的十字架用用么？这个我背不动……” “哼！”朗基努斯不屑地回答了一句，“这可是别人花好多钱帮你特制的呢，别不领情了。” 耶稣正想开口再问，但无奈已经被压得说不出话了。 ———————————————————————————————————————日头升到半空的时候，耶稣终于把十字架背上了山头。一个叫彼拉多的罗马官员主持了行刑。“根据惯例，我要从罪犯里面特赦一个，”他说，“你们是要这个强盗巴拉巴呢，还是要这个耶稣？” “耶稣，耶稣！”保罗他们在底下大喊，希望他们的夫子能免于一死。但他们的声音马上被另一种喊声压下去了。 “巴拉巴，巴拉巴！”一群愤怒的酒徒大喊道，边喊边挥舞着酒瓶。迫于这群人的威势，其他人也跟着一起喊了起来。 “那我要拿耶稣怎么办呢？”彼拉多问。 “钉十字架！钉十字架！”酒鬼们喊。 彼拉多无奈，示意朗基努斯上前把耶稣钉上去。 ———————————————————————————————————————耶稣钉上去以前，向军士们恳求道：“请容我喝一杯临行酒。” 听闻这话，一旁的一个门徒赶紧递上一杯早已准备好的酒。这杯酒就是当时耶稣没来得及喝的毒酒。虽然耶稣觉得在这里服毒自杀很没面子，但好歹不用忍受那疼痛。 朗基努斯暗地里笑了笑，这些伎俩以利早就嘱咐过了。他从杯子旁走过，装作不小心的样子碰翻了那酒。“不好意思啊，”他假惺惺地道歉，然后重新倒了一杯用苦胆调制过的酒给耶稣。 耶稣见毒酒已经倒掉了，就不肯喝了。 军士们把他扒光了，抬到了那个精致的十字架上，用钉子钉住，又按照以利的嘱咐，用海绵吸了强效麻药，绑在苇子上，送给耶稣尝，耶稣尝了麻药，就感觉不到疼痛了，他在十字架上无所事事，一时间也死不掉，就开始效仿那位喝毒酒的先哲，在临死前向在场的观众们大谈人生哲理。 耶稣的十字架本来就异常雄伟，山头又高，俨然居高临下俯瞰着整个耶路撒冷。很多百姓吃了午饭，见到山头上立着这么一个怪东西，都过来看热闹。大家先是很稀奇，竟然会有人挂在那么华丽的十字架上，于是都纷纷凑过来看。然后大家又看到上头的人并非是个恶棍，相反的是个文弱的文化人，于是更好奇了。最后看到这个文化人竟然光着身子，于是一呼百应万人空巷，都来观看这百年奇景。耶稣趁此大谈哲学，倒也博得大家的一阵阵掌声。 与此同时，以利雇佣的人也悄悄在人群中穿梭，发放着木制的小十字架胸章，上面还有耶稣的裸体形象。还有更有甚者在散布流言说，惊讶的时候惊呼“凯撒呀！” 已经不流行了，现在流行喊“耶稣呀！”耶稣还挂在十字架上没断气，已经有近百名群众决定这个星期日就去受洗礼成为基督教徒。 到中午的时候，耶稣已经有点说不动了。他抬头看看天，接引他的天使已经走到半途了，他沉默着，考虑最后一句遗言该说什么。 众人见耶稣沉默了，于是也静下来，等待着他的最后一句话。耶稣四下观看众人，想找点灵感。他突然发现很多人胸前已经别着十字架的小胸章，上面还有自己的半裸体像。谁会事先知道这个事情呢？谁又会对我上十字架那么热心呢？他突然想起以利曾告诉自己他认识一个叫朗基努斯的罗马军士。“这里有一位朗基努斯军爷没有？”他问。 朗基努斯走过来，“谁叫我？” 耶稣顿时明白了，在天使降临前的一瞬，他愤怒地用拿撒勒的土话向远方大喊：“以利！以利！拉马撒巴各大尼！” 随后，天使的翅膀就覆盖了耶稣基督的灵魂。 ———————————————————————————————————————自此，基督的事迹就在世间流传，使徒们的行为也为众人传颂。遵照耶稣的意愿，马太四人动笔记录他的生平，他们走访耶稣生前的朋友们以收集信息。他们向以利提出的第一个问题就是：“耶稣死前喊的那句话是什么意思？” 以利笑着回答：“那句话的意思是，‘我的神，我的神，为什么离弃我？’” 四个人用狐疑的眼光看着他。 以利回答：“好吧，好吧，在拿撒勒土语里，那句话的意思是，‘以利！以利！咱俩没完！’” “没完什么？”路加问以利，而后者则笑而不答了。 “算了，就以您前一个回答为准吧。”路加无奈地说。 ———————————————————————————————————————以利活了84岁，日子满足而死。当他来到天堂后，又重新听到了上帝的声音。 “以利，你干得真漂亮！” “谢谢，主，不知道我主的宗教能延续多少年？” “至少两千年，比我的要求高了一倍。在你的帮助下，我的独子终于及格了。” “谢谢，主，愿我主的名传遍天下。” “只有半个天下而已。” “半个天下？另半个是谁的？” “另半个是默罕默德的伊斯兰教的。” “不可能！基督教是我用商业理论一手策划的，默罕默什么的，他有这本事创造一个匹敌的宗教吗？” “他没有，但他也有一个朋友帮他，那人你也认识，是你的同学，叫穆萨。” “穆萨？不可能！这个呆子一问三不知，根本什么都没学会，怎么可能同我相比。” “蠢材！最后一节课你没有去上，他去了，而且学得很好。” “最后一节课那么重要？” “废话！他回来后，教穆罕默德宣称伊斯兰教才是基督教的正源，耶稣的一切活动都是为默罕默德的降临做准备的，耶稣是伊斯兰教的先知。他剽窃了你的一切成就，踩在你的肩膀上获取了成功。” “我主……”以利无言以对。 上头安慰他说，“别自责了，你的表现很好了，不过……”祂离去前，最后撂下了一句话：“我早告诉你了，上最后一节课的是韩国人。”
2009年4月20日星期一
The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification
Identification of components present in biological complexesrequires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to designa general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool thatallows rapid purification under native conditions of complexes,even when expressed at their natural level. Prior knowledge ofcomplex composition or function is not required. The TAP methodrequires fusion of the TAP tag, either N- or C-terminally, to thetarget protein of interest. Starting from a relatively small numberof cells, active macromolecular complexes can be isolated andused for multiple applications. Variations of the method to specifically purify complexes containing two given components or tosubtract undesired complexes can easily be implemented. The TAPmethod was initially developed in yeast but can be successfullyadapted to various organisms. Its simplicity, high yield, and wideapplicability make the TAP method a very useful procedure forprotein purification and proteome exploration.
The sequencing of complete genomes of several organisms provides an exceptional opportunity to analyze thedifferent functions governed by their genes. Insightsinto these complex biological systems can be gained byanalysis of gene regulatory networks and by determining the identity, modification, and expression levels ofencoded proteins as well as by defining interactionsexisting among proteins (proteomic analyses). Large-scale two-hybrid screening has been used for this latter purpose (1–3). However, false-positive and false-negative results, the lack of information about stoichiometry,and the limited set of conditions testable make it desirable to use additional strategies to easily detect protein interactions.
Biochemical purification of proteins in combinationwith mass spectrometry allows identification of interacting partners. This strategy is becoming an importanttool to define relations existing among gene products(4, 5). Currently, ,100 fmol of a protein can be detectedand identified by mass spectrometry, allowing rapidcharacterization of any protein present in a complexmixture, provided that the target complex is sufficientlypurified in reasonable quantity. Identification of proteins by mass spectrometry is currently facilitated forseveral organisms by the availability of complete genomic sequences. The current limiting step in protein complex characterization appears therefore to be proteinpurification rather than protein identification. Eachprotein has unique properties, which can be exploitedfor its purification (6). This makes it, however, impossi-ble to design a general purification strategy valid forall cases. A generic purification protocol is thereforedesirable to allow routine and possibly automated protein complex purification for proteome analysis. Thefusion of tags, peptides, or protein domains to proteintargets appeared best suited toward this goal. Aftercomparative testing of several tags, we have recentlydeveloped a new tag, the tandem affinity purification(TAP) tag, and we have optimized a procedure, the TAPmethod, for the native purification of protein complexes(7). This strategy allows for fast purification with highyield of protein complexes under standard conditions.Ultimately, the purified complex can be used for protein identification, functional, orstructuralstudies. Furthermore, variations on the original strategy, including the use of C- or N-terminal tags, the use of a split tag, and/or the use of a subtraction step can easily be developed. These various aspects are described below starting with a presentation of the basic TAP method from gene tagging to protein analysis. We also present several applications of the method and discuss different variations from the original protocol and potential problems. General guidelines useful for various organisms are given; however, as the TAP method was developed with yeast, emphasis is given to applications in this organism. Detailed protocols and latest developments can also be found on our web site(http://www.emblheidelberg.de/ExternalInfo/seraphin/TAP.html).
METHODS
1.Over view of the TAP Method and the TAP Tag
The TAP method involves the fusion of the TAP tag (seebelow) to the target protein and the introduction of the construct into the host cell or organism. For optimal results, it is preferable to maintain expression of the fusion protein at, or close to, its natural level. Indeed, over expression of the protein often induces its association with nonnatural partners (heat shock proteins, proteasome; Ref. (8)). Cell extracts are prepared and the fusion protein as well asassociated partners is recovered by two specific affinity purification/elution steps. The material recovered can be analyzed in several ways. For protein complex characterization, proteins are concentrated, and eventually fractionated on a denaturing gel, before identification by mass spectrometry. (Alternatively, Edman degradation or Western blot may be used.) Because the various TAP purification steps are performed in a gentle native manner, purified complexes may also be tested for their activities or used in structural analysis.
The TAP tag consists of two IgG binding domains of Staphylococcus aureus protein A (ProtA) and a calmodulin binding peptide (CBP) separated by a TEV protease cleavage site. Originally, a C-terminal TAP tag was described (7) (Fig. 1A). We have now also generated an N-terminal TAP tag (Fig. 1A, seebelow). Note that the relative order of the modules of the TAP tag are inversed in the two tags because the ProtA module needs to be located at the extreme N or C terminus of the fusion protein. Both affinity tags have been selected for highly efficient recovery of proteins present at low concentration. ProtA binds tightly to an IgG matrix, requiring the use of the TEV protease to elute material under native conditions (Fig. 1B). The eluate of this first affinity purification step is then incubated with calmodulin coated beads in the presence of calcium. After washing, which removes contaminants and the TEV protease remaining after the first affinity selection, the bound material is released under mild conditions with EGTA (Fig. 1B). Optimized conditions have been developed for the generic use of the TAP strategy (see below). The TAP tag is, however, very tolerant to buffer conditions and changes can easily be implemented to optimize recovery of specific complexes.
2.Tagging the Target Protein with the TAP Tag
The choice of the strategy for fusing the TAP tag to the target protein depends on the methods available to introduce recombinant nucleic acids into the corresponding cell or organism. One should also keep in mind that strong over expression of the target protein is not preferable except if one is interested in producing large amounts of this protein by itself. Indeed, protein overexpression may often lead to the formation of nonspecific and/or nonnatural protein interactions with host proteins (8). This should be avoided if one wants to identify the structure, composition, and/or activity of acomplex. The TAP tag has been specifically designed to allow recovery of proteins expressed at their low natural levels. Usually, standard DNA cloning procedures can be used to introduce the N- or C- terminal TAP tag in-frame with the coding region of the protein of interest in an appropriate expression vector. For this purpose, unique
restriction sites present upstream and downstream of the N- and C-terminal TAP cassettes are available (Fig. 2A). The recombinant vector can then be transiently or stably introduced into recipient cells ororganisms. Optimally, the tagged construct should be used to replace the endogenous wild-type gene. However, depending on the organism analyzed, this is not always possible and often time consuming (e.g., construction of transgenic mice).
The high efficiency of homologous recombination in yeast bypasses the need to construct a plasmid to fuse the TAP tag to the protein of interest. Polymerase chain reaction (PCR) fragments can indeed be used to integrate the TAP tag directly in the genome (9,10). We routinely prefer to use the C- terminal TAP tag for this purpose as this maintains expression of the target protein under the control of its natural promoter. However, some proteins undergo loss of function when a peptide is added to its C-terminus. While from our experience this is not very frequent (about 5% of fusions), it is worthwhile to introduce the TAP tag into both haploid and diploid cells in parallel to test this possibility. For cases where problems are encountered with the C-terminal TAP tag, we have designed a strategy that allows genomic fusion of an N-terminal TAP tag to the protein of interest while maintaining its expression under control of the endogenous promoter (see variations of the TAP method below).
The two plasmids constructed in our laboratory to introduce the C-terminal TAP tag into the yeast genome differ by the presence of either a URA3 or a TRP1 marker from Kluyveromyces lactis adjacent to the TAPc assette(Fig. 2A, pBS1479 and pBS1539, respectively). Primers containing a region of similarity to the yeast genome (40-50 nt long) and a constant priming regio (Fig. 2B) are synthesized. Primer A hybridizes at the 5' end of the CBP coding sequence and primer B in the vector downstream of the selection marker. Primer A should be carefully designed such that the last C-terminal residue of the target protein gets fused in-frame to the TAP tag. These primers are used to amplify by PCR the TAP tag from plasmid pBS1479 or pBS1539. The PCR product is extracted with phenol/chloroform/isoamylalcohol, precipitated, and used to transform haploid and diploid yeast cells (11,12). Correct integration of the cassette is verified by PCR and/or Southern blot (13,14). To check for expression of the tagged protein, Western blot is used. Briefly, the cellular pellets corresponding to 1.5 ml of cellculture are vortexed 3×30s with30 μl siliconized glass beads and 100 μl of SDS-PAGE loading buffer. Samples are boiled, vortexed once more, and loaded directly on an SDS-Cpolyacrylamid gel. Western blots are developed with a peroxidase-antiperoxidase complex (PAP, Sigma P-2026) that detects ProtA. However, one should remember that this strategy might not be sufficiently sensitive if the target protein is expressed at a very low level.
3. Extract preparation
Various extraction procedures can be used to prepare extracts from cells or organisms expressing the target protein fused to the TAP tag. The choice of the appropriate extract preparation procedure will depend on the target protein and on prior experience in the field that can be found in the literature. Cell fractionation and/or tissue dissection can facilitate purification by providing a preenrichment step or can be used to assay specifically protein complex composition in various tissues or cell compartments. In general, however, it is advisable to check, by detecting the ProtA moiety of the TAP tag by Western blot, whether extraction is efficient and if the TAP tag is not degraded under these specific conditions.
For yeast, we recommend the following standard procedure that has been extensively used in our laboratory. However, this method is unlikely to be optimal for all proteins and alternative protein extraction methods may be used (see variations in the purification protocol below). Extracts are routinely prepared from 2 liters of yeast cells grown to late log phase (OD600～2–3). Cell pellets are washed once with water and pelleted againin a 50-ml polypropylene tube (Falcon). The packed cell volume (PCV) is measured and the tube is frozen with liquid nitrogen. Frozen cell pellets may be stored at
The sequencing of complete genomes of several organisms provides an exceptional opportunity to analyze thedifferent functions governed by their genes. Insightsinto these complex biological systems can be gained byanalysis of gene regulatory networks and by determining the identity, modification, and expression levels ofencoded proteins as well as by defining interactionsexisting among proteins (proteomic analyses). Large-scale two-hybrid screening has been used for this latter purpose (1–3). However, false-positive and false-negative results, the lack of information about stoichiometry,and the limited set of conditions testable make it desirable to use additional strategies to easily detect protein interactions.
Biochemical purification of proteins in combinationwith mass spectrometry allows identification of interacting partners. This strategy is becoming an importanttool to define relations existing among gene products(4, 5). Currently, ,100 fmol of a protein can be detectedand identified by mass spectrometry, allowing rapidcharacterization of any protein present in a complexmixture, provided that the target complex is sufficientlypurified in reasonable quantity. Identification of proteins by mass spectrometry is currently facilitated forseveral organisms by the availability of complete genomic sequences. The current limiting step in protein complex characterization appears therefore to be proteinpurification rather than protein identification. Eachprotein has unique properties, which can be exploitedfor its purification (6). This makes it, however, impossi-ble to design a general purification strategy valid forall cases. A generic purification protocol is thereforedesirable to allow routine and possibly automated protein complex purification for proteome analysis. Thefusion of tags, peptides, or protein domains to proteintargets appeared best suited toward this goal. Aftercomparative testing of several tags, we have recentlydeveloped a new tag, the tandem affinity purification(TAP) tag, and we have optimized a procedure, the TAPmethod, for the native purification of protein complexes(7). This strategy allows for fast purification with highyield of protein complexes under standard conditions.Ultimately, the purified complex can be used for protein identification, functional, orstructuralstudies. Furthermore, variations on the original strategy, including the use of C- or N-terminal tags, the use of a split tag, and/or the use of a subtraction step can easily be developed. These various aspects are described below starting with a presentation of the basic TAP method from gene tagging to protein analysis. We also present several applications of the method and discuss different variations from the original protocol and potential problems. General guidelines useful for various organisms are given; however, as the TAP method was developed with yeast, emphasis is given to applications in this organism. Detailed protocols and latest developments can also be found on our web site(http://www.emblheidelberg.de/ExternalInfo/seraphin/TAP.html).
METHODS
1.Over view of the TAP Method and the TAP Tag
The TAP method involves the fusion of the TAP tag (seebelow) to the target protein and the introduction of the construct into the host cell or organism. For optimal results, it is preferable to maintain expression of the fusion protein at, or close to, its natural level. Indeed, over expression of the protein often induces its association with nonnatural partners (heat shock proteins, proteasome; Ref. (8)). Cell extracts are prepared and the fusion protein as well asassociated partners is recovered by two specific affinity purification/elution steps. The material recovered can be analyzed in several ways. For protein complex characterization, proteins are concentrated, and eventually fractionated on a denaturing gel, before identification by mass spectrometry. (Alternatively, Edman degradation or Western blot may be used.) Because the various TAP purification steps are performed in a gentle native manner, purified complexes may also be tested for their activities or used in structural analysis.
The TAP tag consists of two IgG binding domains of Staphylococcus aureus protein A (ProtA) and a calmodulin binding peptide (CBP) separated by a TEV protease cleavage site. Originally, a C-terminal TAP tag was described (7) (Fig. 1A). We have now also generated an N-terminal TAP tag (Fig. 1A, seebelow). Note that the relative order of the modules of the TAP tag are inversed in the two tags because the ProtA module needs to be located at the extreme N or C terminus of the fusion protein. Both affinity tags have been selected for highly efficient recovery of proteins present at low concentration. ProtA binds tightly to an IgG matrix, requiring the use of the TEV protease to elute material under native conditions (Fig. 1B). The eluate of this first affinity purification step is then incubated with calmodulin coated beads in the presence of calcium. After washing, which removes contaminants and the TEV protease remaining after the first affinity selection, the bound material is released under mild conditions with EGTA (Fig. 1B). Optimized conditions have been developed for the generic use of the TAP strategy (see below). The TAP tag is, however, very tolerant to buffer conditions and changes can easily be implemented to optimize recovery of specific complexes.
2.Tagging the Target Protein with the TAP Tag
The choice of the strategy for fusing the TAP tag to the target protein depends on the methods available to introduce recombinant nucleic acids into the corresponding cell or organism. One should also keep in mind that strong over expression of the target protein is not preferable except if one is interested in producing large amounts of this protein by itself. Indeed, protein overexpression may often lead to the formation of nonspecific and/or nonnatural protein interactions with host proteins (8). This should be avoided if one wants to identify the structure, composition, and/or activity of acomplex. The TAP tag has been specifically designed to allow recovery of proteins expressed at their low natural levels. Usually, standard DNA cloning procedures can be used to introduce the N- or C- terminal TAP tag in-frame with the coding region of the protein of interest in an appropriate expression vector. For this purpose, unique
restriction sites present upstream and downstream of the N- and C-terminal TAP cassettes are available (Fig. 2A). The recombinant vector can then be transiently or stably introduced into recipient cells ororganisms. Optimally, the tagged construct should be used to replace the endogenous wild-type gene. However, depending on the organism analyzed, this is not always possible and often time consuming (e.g., construction of transgenic mice).
The high efficiency of homologous recombination in yeast bypasses the need to construct a plasmid to fuse the TAP tag to the protein of interest. Polymerase chain reaction (PCR) fragments can indeed be used to integrate the TAP tag directly in the genome (9,10). We routinely prefer to use the C- terminal TAP tag for this purpose as this maintains expression of the target protein under the control of its natural promoter. However, some proteins undergo loss of function when a peptide is added to its C-terminus. While from our experience this is not very frequent (about 5% of fusions), it is worthwhile to introduce the TAP tag into both haploid and diploid cells in parallel to test this possibility. For cases where problems are encountered with the C-terminal TAP tag, we have designed a strategy that allows genomic fusion of an N-terminal TAP tag to the protein of interest while maintaining its expression under control of the endogenous promoter (see variations of the TAP method below).
The two plasmids constructed in our laboratory to introduce the C-terminal TAP tag into the yeast genome differ by the presence of either a URA3 or a TRP1 marker from Kluyveromyces lactis adjacent to the TAPc assette(Fig. 2A, pBS1479 and pBS1539, respectively). Primers containing a region of similarity to the yeast genome (40-50 nt long) and a constant priming regio (Fig. 2B) are synthesized. Primer A hybridizes at the 5' end of the CBP coding sequence and primer B in the vector downstream of the selection marker. Primer A should be carefully designed such that the last C-terminal residue of the target protein gets fused in-frame to the TAP tag. These primers are used to amplify by PCR the TAP tag from plasmid pBS1479 or pBS1539. The PCR product is extracted with phenol/chloroform/isoamylalcohol, precipitated, and used to transform haploid and diploid yeast cells (11,12). Correct integration of the cassette is verified by PCR and/or Southern blot (13,14). To check for expression of the tagged protein, Western blot is used. Briefly, the cellular pellets corresponding to 1.5 ml of cellculture are vortexed 3×30s with30 μl siliconized glass beads and 100 μl of SDS-PAGE loading buffer. Samples are boiled, vortexed once more, and loaded directly on an SDS-Cpolyacrylamid gel. Western blots are developed with a peroxidase-antiperoxidase complex (PAP, Sigma P-2026) that detects ProtA. However, one should remember that this strategy might not be sufficiently sensitive if the target protein is expressed at a very low level.
3. Extract preparation
Various extraction procedures can be used to prepare extracts from cells or organisms expressing the target protein fused to the TAP tag. The choice of the appropriate extract preparation procedure will depend on the target protein and on prior experience in the field that can be found in the literature. Cell fractionation and/or tissue dissection can facilitate purification by providing a preenrichment step or can be used to assay specifically protein complex composition in various tissues or cell compartments. In general, however, it is advisable to check, by detecting the ProtA moiety of the TAP tag by Western blot, whether extraction is efficient and if the TAP tag is not degraded under these specific conditions.
For yeast, we recommend the following standard procedure that has been extensively used in our laboratory. However, this method is unlikely to be optimal for all proteins and alternative protein extraction methods may be used (see variations in the purification protocol below). Extracts are routinely prepared from 2 liters of yeast cells grown to late log phase (OD600～2–3). Cell pellets are washed once with water and pelleted againin a 50-ml polypropylene tube (Falcon). The packed cell volume (PCV) is measured and the tube is frozen with liquid nitrogen. Frozen cell pellets may be stored at
2009年4月18日星期六
Protocol and procedure for silver staining（蛋白银染）
GE Healthcare
Instructions 71-7177-00 AL
PlusOne Silver Staining Kit, Protein
General information
Wear gloves at all times. Perform all steps at 21 to 25°C with constant gentle agitation on a shaker.
The quality of the water used for making up the reagents and for washing the gel will affect the staining result. For best results use water with a conductivity of 5 MΩ or more (e.g. Milli-Q™ water).
Freshly made solutions (not older than 24 hours) give best results.
Precautions: The chemicals in this kit should not be discarded via public waste water systems. Please dispose of these chemicals properly. Consult your local regulations for more information.Read the warning text on the label of each bottle and packet.
ExcelGel SDS, ExcelGel XL SDS 12-14 and mini slab gels 0.5 mm
Chemicals required
• Chemicals included in this PlusOne Silver Staining Kit, Protein
• Ethanol
• Glacial acetic acid
• Glycerol (87% w/w) (1000 ml) (Code No. 17-1325-01)
Reagents
Make up the following reagent solutions using the chemicals listed above.
250 ml solution is needed per gel and step except for ExcelGel XL SDS 12-14, which requires 400 ml per gel and step and for Mini slab gels which require 125 ml solution per gel and step.
Note: Glutardialdehyde and formaldehyde should be added immediately before use.
Instructions 71-7177-00 AL
PlusOne Silver Staining Kit, Protein
General information
Wear gloves at all times. Perform all steps at 21 to 25°C with constant gentle agitation on a shaker.
The quality of the water used for making up the reagents and for washing the gel will affect the staining result. For best results use water with a conductivity of 5 MΩ or more (e.g. Milli-Q™ water).
Freshly made solutions (not older than 24 hours) give best results.
Precautions: The chemicals in this kit should not be discarded via public waste water systems. Please dispose of these chemicals properly. Consult your local regulations for more information.Read the warning text on the label of each bottle and packet.
ExcelGel SDS, ExcelGel XL SDS 12-14 and mini slab gels 0.5 mm
Chemicals required
• Chemicals included in this PlusOne Silver Staining Kit, Protein
• Ethanol
• Glacial acetic acid
• Glycerol (87% w/w) (1000 ml) (Code No. 17-1325-01)
Reagents
Make up the following reagent solutions using the chemicals listed above.
250 ml solution is needed per gel and step except for ExcelGel XL SDS 12-14, which requires 400 ml per gel and step and for Mini slab gels which require 125 ml solution per gel and step.
Note: Glutardialdehyde and formaldehyde should be added immediately before use.
Staining procedure
All steps should be performed with gentle shaking of the staining tray.
1 Fixation: 30 min.
Soak the gel in fixing solution for 30 minutes.
2 Sensitizing: 30 min.
Remove the solution. Add sensitizing solution and leave shaking for at least 30 minutes.
3 Washing: 3 x 5 min.
Remove the sensitizing solution. Add distilled water and wash three times for 5 minutes each time.
4 Silver reaction: 20 min.
Add silver solution and leave shaking for 20 minutes.
5 Washing: 2 x 1 min.
Remove the silver solution. Rinse twice in distilled water for one minute each time.
6 Developing: 2 to 5 min.
Add developing solution and leave shaking for 2 to 5 minutes. Transfer the gel to stopping solution when the bands / spots have reached desired intensity.
7 Stopping: 10 min.
Leave gel shaking in stopping solution for 10 minutes.
8 Washing: 3 x 5 min.
Remove the stop solution. Add distilled water and wash three times for 5 minutes each time.
9Preserving:
20 min. (plastic-backed gels)
2 x 30 min. (gels not supported on plastic films)
Add preserving solution and leave shaking for 20 minutes for plastic backed gels. For gels not supported on plastic films, shake for 30 minutes, pour off the solution, add fresh and shake for a further 30 minutes.
10 Drying: Overnight
Put the gel on a glass plate and wrap it in Cellophane sheet. Leave the gel to dry overnight at room temperature. Do not put the gel in a heating cabinet (the silver stain bleaches at elevated temperatures).
对旅行者的50句忠告
一、 不要接近峨眉山的和尚。
二、 不要接近崂山的道士。
三、 不要买九寨沟的牦牛肉。
四、 不要买三峡船上的玉器珠宝。
五、 不要在西双版纳参加“抢亲”游戏。
六、 不要在西安的古玩一条街购买古物。
七、 不要在大理购买所谓“老乡”的便宜珠宝。
八、 不要在阳朔的酒吧里消费。
九、 不要招惹泰山景区的当地人。
十、 不要参观少林寺时走“捷径”。
十一、 不要逛十三陵“***”玉石店。
十二、 不要单独去海南旅游
十三、 不要在坝上草原骑马。
十四、 不要在吐鲁番买葡萄。
十五、 不要在深圳中英街购买任何物品。
十六、 不要参加北京当地的长城一日游。
十七、 不要在张家界住便宜小旅馆。
十八、 不要在井冈山为“老区建设”捐款。
十九、 不要在杭州的娱乐场所消费。
二十、 不要在苏州的茶楼喝茶。
二十一、 不要在丰都鬼城照“免费”像。
二十二、 不要在北海乘坐摩托艇。
二十三、 不要在北戴河吃海鲜。
二十四、 不要在三亚海边接近小商贩。
二十五、 不要随导游在呼伦贝尔草原上吃烤全羊。
二十六、 不要在庐山乘个体出租车上山。
二十七、 不要在宏村的路上坐出租车。
二十八、 不要去黄山让人“免费带路”。
二十九、 不要在千岛湖码头的排档吃鱼。
三十、 不要参观乐山景区周边的付费景点。
三十一、 不要在郑州黄河游览区骑马。
三十二、 不要到恒山算命。
三十三、 不要随旅游团环游青海湖。
三十四、 不要买神农架的土特产。
三十五、 不要在敦煌的夜市吃地摊饭。
三十六、 不要在花果山的海鲜一条街吃饭。
三十七、 不要随导游逛清明上河园。
三十八、 不要在南京做“免费美容”。
三十九、 不要在上海外滩让人给你照数码像。
四十、 不要买“便宜票”看黄果树大瀑布。
四十一、 不要随导游在香港买名表和珠宝。
四十二、 不要投大钱在澳门赌博。
四十三、 不要在威海韩国城购买没经狠杀价的商品。
四十四、 不要在丽江洗桑拿浴。
四十五、 不要到五台山的五爷庙烧香。
四十六、 不要在平遥摸“鱼洗”。
四十七、 不要在武夷山景区买茶叶。
四十八、 不要在白洋淀景区买鸭蛋。
四十九、 不要在乌镇让道士“免费看相”。
五十、 不要去太阳岛坐“热心人”介绍的船
二、 不要接近崂山的道士。
三、 不要买九寨沟的牦牛肉。
四、 不要买三峡船上的玉器珠宝。
五、 不要在西双版纳参加“抢亲”游戏。
六、 不要在西安的古玩一条街购买古物。
七、 不要在大理购买所谓“老乡”的便宜珠宝。
八、 不要在阳朔的酒吧里消费。
九、 不要招惹泰山景区的当地人。
十、 不要参观少林寺时走“捷径”。
十一、 不要逛十三陵“***”玉石店。
十二、 不要单独去海南旅游
十三、 不要在坝上草原骑马。
十四、 不要在吐鲁番买葡萄。
十五、 不要在深圳中英街购买任何物品。
十六、 不要参加北京当地的长城一日游。
十七、 不要在张家界住便宜小旅馆。
十八、 不要在井冈山为“老区建设”捐款。
十九、 不要在杭州的娱乐场所消费。
二十、 不要在苏州的茶楼喝茶。
二十一、 不要在丰都鬼城照“免费”像。
二十二、 不要在北海乘坐摩托艇。
二十三、 不要在北戴河吃海鲜。
二十四、 不要在三亚海边接近小商贩。
二十五、 不要随导游在呼伦贝尔草原上吃烤全羊。
二十六、 不要在庐山乘个体出租车上山。
二十七、 不要在宏村的路上坐出租车。
二十八、 不要去黄山让人“免费带路”。
二十九、 不要在千岛湖码头的排档吃鱼。
三十、 不要参观乐山景区周边的付费景点。
三十一、 不要在郑州黄河游览区骑马。
三十二、 不要到恒山算命。
三十三、 不要随旅游团环游青海湖。
三十四、 不要买神农架的土特产。
三十五、 不要在敦煌的夜市吃地摊饭。
三十六、 不要在花果山的海鲜一条街吃饭。
三十七、 不要随导游逛清明上河园。
三十八、 不要在南京做“免费美容”。
三十九、 不要在上海外滩让人给你照数码像。
四十、 不要买“便宜票”看黄果树大瀑布。
四十一、 不要随导游在香港买名表和珠宝。
四十二、 不要投大钱在澳门赌博。
四十三、 不要在威海韩国城购买没经狠杀价的商品。
四十四、 不要在丽江洗桑拿浴。
四十五、 不要到五台山的五爷庙烧香。
四十六、 不要在平遥摸“鱼洗”。
四十七、 不要在武夷山景区买茶叶。
四十八、 不要在白洋淀景区买鸭蛋。
四十九、 不要在乌镇让道士“免费看相”。
五十、 不要去太阳岛坐“热心人”介绍的船
2009年4月17日星期五
2009年4月16日星期四
两招让你的U盘从坟墓中苏醒
第一招：系统高级格式化…… 点击开始=》运行=》然后输入cmd，在弹出窗口再输入 chkdsk x:/f(x代表u盘盘符，x前有空格哦)
第二招：量产工具…… 这个可是要下载软件的！不过放心都只有几Mb而已！重点是都能用都是免费的！ 但是要注意！要对好主控芯片！！ U盘就只有那么两块芯片而已！（内存、主控）小的就是！ 我的这个是“MW6208E”
我下了一个量产工具（上百度搜：“MW6208E”就可以了） 接下来的就是按一下就行了！！记住哦！软件里的东西不要随意改！ 默认的就行了！这可是终极方法了！不行就真的扔了它吧！
第二招：量产工具…… 这个可是要下载软件的！不过放心都只有几Mb而已！重点是都能用都是免费的！ 但是要注意！要对好主控芯片！！ U盘就只有那么两块芯片而已！（内存、主控）小的就是！ 我的这个是“MW6208E”
我下了一个量产工具（上百度搜：“MW6208E”就可以了） 接下来的就是按一下就行了！！记住哦！软件里的东西不要随意改！ 默认的就行了！这可是终极方法了！不行就真的扔了它吧！
2009年4月15日星期三
裏技--生活贴士和技巧
“裏技”在日语中是聪明的生活贴士和技巧的意思。
1.商店的读卡器没能成功的读取磁条：把卡用一层塑料袋包了起来试一遍
2.一片面包捡起厨房地板上的碎玻璃
3.旅行时把养的植物放在湿尿布上来为它们浇水
4.袋里的手机不用也快速的消耗电量：原因之一可能是你的口袋过于温暖了。手机电池在凉快的时候确实使用时间要长一点，如果想让它凉快一些，就把手机放在钱包里或别在腰带上。这方法也可以用在出门却发现没带充电器的时候，关掉电话并把电话放在宾馆的冰箱里可以减慢电池自然放电的速度。
5.汽车遥控钥匙不能越过停车场打开车门:把钥匙链上的金属钥匙按在下巴上再按下开锁键，这个技巧把你的头作为天线。当钥匙链和你的头部连接起来时形成了一个电容。你头部里的液体把它变成的一个良好导体。虽然不是非常完美，但也足以起作用。用你的头可以使遥控距离增大几辆车的范围。
6.打印机墨盒在快完成一项重要任务时干了：拿下墨盒并用电吹风吹两三分钟，然后趁热把它装回去再试一遍。电吹风的热量把粘稠的墨水加热，帮助它流过墨盒里微小的喷嘴，当墨盒快用完时，这些喷嘴常常会被干墨堵塞，所以帮助墨水流动能让更多墨水流出喷嘴。电吹风能在打印机声明墨盒已空后多打出几页纸。
7.手机掉进了马桶：为了防止短路对脆弱的内部结构造成损伤要立刻拿下电池。然后小心地用毛巾包住手机，并放入装满生米的罐头瓶里。这和在盐盒里放几粒米防潮道理一样。米在化学上对水有很强的亲和力——意思是米的分子对水分子有磁性一般的吸引力，使它们蒸发进入米里而不是在手机里凝结成水珠。这是“禁止食用”干燥剂的低技术版。和那些手机盒子里为了在运输和贮藏过程中防潮的小包有同样功效。
8.家中的Wi-Fi路由器覆盖不了房子的另一头:别急着买更多的接入设备来扩展网络。你可以用厨房里的东西做个六英寸的被动无线电波反射器，比如烹饪铝箔。像电视卫星天线一样反射电波的金属小曲面放在你的Wi-Fi路由器后边。它能把路由器的能量汇聚到一个方向——朝着房子的另一端——而不是分散到整个环形空间。无需线缆，无需电池，甚至无需专业知识，用它就能轻松地把网络覆盖距离加倍。
9.需要清洁一张跳音的CD或DVD:作为个单身汉又没有那么有女人味的清洁剂怎么办？用伏特加或漱口水把抹布浸湿吧。酒精是强力溶剂，对碟片上的指纹和灰尘有绝佳效果。药品抽屉里5美元一瓶的李施德林漱口水和75美元的DVD清洁液有同样的效果。而且，用苏联红牌威士忌清洁你的“Lost Weekend”不是比用光盘清洁套装更有男人味么？
10.手机自带的闪光灯太亮了：搞得相片一片白的话，用胶带往闪光灯前面贴一小块纸。试试不同颜色不同厚度的纸来把相片从过曝调整到更令人舒适的晚间光影效果。
11.电脑硬盘崩溃了不能读出数据：别急着扔掉它。先把它在冷冻室放一晚。这个技巧是真的且经过验证的，尽管是最后手段，对某些非致命硬盘问题的恢复技术。许多硬盘问题是因为长时间使用之后的部件接合不紧密造成的硬盘不可读。降温使得硬盘内部的金属和塑料部件接合的更加紧密。将硬盘拿出冷冻室，等它回到室温就能使那些部件重新连接起来。这可以让不工作的部件重新启动，或至少让即将损坏的电子元件能维持到恢复出你的重要数据。
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