pipet-80

日语外来语 ho－ ru pi pe 促音 to 是什么意思？

移液器的选择，一般注意从以下几个方面进行考虑：

对于绝大多数用户而言，购买之前检测产品性能既有难度又无必要。因此，主要还是依据制造厂商提供的技术数据。但在这里还是要说明两点：

其一，不要轻易相信卖家的口头承诺，一定要查阅制造商提供的书面材料；

其二，在全球移液器市场上影响较大的品牌，，其提供的技术数据可信度更高。

这一方面，主要取决于移液器所用的材料。对于外壳，应当有较高的耐冲击性、耐腐蚀性和较低的导热性（如PVDF材质）。对于活塞，目前市场上主要有不锈钢、陶瓷和塑料三种材质。不锈钢机械性能好、寿命长，只是不太适合用于强酸强碱的移液；陶瓷则有很高的耐腐蚀性，但机械性能较差。

梅特勒-托利多有Pipet-Lite XLS+手动单道移液器、Pipet-Lite XLS+手动多通道移液器、Pipet-Lite XLS间距可调手动多道移液器、Pipet-Lite PL+ 移液器、Pipet-One 移液器，还有E4 XLS+ 单道电动移液器、E4 XLS+ 多道电动移液器、E4 XLS间距可调多道电动移液器，满足不同场景的应用需求。

测试方面的英语翻译，请指教！

whole pipet

前端细，中间粗的有容量表示线的化学吸管。用来吸取定量的液体。如果学化学的一看就知道。

猪圆环病毒2型与猪附红细胞体混合感染的诊断与防治

注：如果来自标准溶液A的污点变暗，这种情况预示烤盘在烤炉放置时间过长，应该准备第二个盘子。

甲醇

内部标准溶液：用吸管将正丙醇1.0毫升注入一个100毫升量瓶，用吡啶（氮杂苯）稀释到一定量并混合。把5.0毫升这种溶液的倒转移到一个500毫升量瓶。用内部标准溶液稀释到一定量并混合。把10毫升的这种溶液转移到一个100毫升量瓶，用内部标准溶液稀释到一定量并混合。

样本溶液：，准确秤过后将样本约2克转移到一个10毫升量瓶，用内部标准溶液稀释到一定量并混合。

程序（参阅色谱法，附录IIA）：使用装有氢火焰温度检测器的相配的气相色谱系统和一根用80-网孔到100-网孔的聚苯乙烯型色谱固定相包裹的2.1-m乘4-mm(标识)玻璃棒或等同代用工具。把玻璃棒调整到150度（恒温），插入物到200度，检验器到250度。用氦气作为携带气体，调整到流速20 毫升/秒。把1-μL部分标准溶液注入气相色谱仪获得色谱，测量生成物高峰的面积。（甲醇滞留时间大约为分钟。）重复注射而用的相对的标准溶液不多于2.0%。计算标准溶液的平均高峰。如前，把1-μL部分标准溶液注入气相色谱仪，测量由甲醇产生的各种高峰的面积。计算平均面积，用下述方程式确定选取的样本甲醇分离部分的百分比。

其中元素钌是甲醇峰面积的系数与从样本溶液获取的内部标准溶液系数之比：甲醇峰面积系数中的Rs（供选择：重返大气装置、反转信号、继电器式选择器）与从标准溶液获取的内部标准溶液相比；因子0.158等于标准中的甲醇值 X 稀释因子 X 甲醇密度 X 100%；Ws（操作取样）是样本的重量，计以克数。

Native-PAGE与SDS-PAGE的区别是什么？

猪圆环病毒2型与猪附红细胞体混合感染的诊断与防治。

税光伟 韩旭 张成伟 洪洋 魏攀 指导老师：赵光伟。

西南大学荣昌校区动物医学系，荣昌 402460。

摘要:随着我国养猪业集约化程度的提高，生猪和种猪跨地域运输越来越频繁，使得猪群传染病的发生越来越复杂。在很多猪场，都同时存在多种病原的混合感染。2009年1月四川隆昌某猪场送检病猪3头。该场中部分猪只陆续发生高热、皮肤发绀、呼吸困难等症状，病程一般持续5～7天，发病猪一般以死亡为转归。不同年龄段的猪群中均有不同程度的发生，临床表现基本相似。通过临床症状和病理剖检，疑似猪蓝耳病病毒（PRRSV）、猪圆环病毒2型（PCV-2）、猪附红细胞体（E.suis）混合感染。据此笔者在西南大学荣昌校区动物疾病快速诊断中心做了确诊实验。

关键词:圆环病毒2型 附红细胞体 混合感染 综合防治 。

Diagnosis and Treatment。

of Porcine circovirus type 2 mixed infection Eperythrozoon 。

Shui Guang Wei

Southwest China Normal University of Rongchang campus veterienary medicine department, Rongchang 402460, China。

Abstract: Along with our country pig-breeding industry intensification degree's enhancement, the live pig and the kind of pig cross region transportation is getting more and more frequent, causes the pig group infectious disease's occurrence to be getting more and more complex. In many pig farms, simultaneously has many kinds of causes of disease the mixed infections. In January, 2009 Sichuan Longchang one pig farm deliver examines the sickness pig 3 heads. This partial pigs only one after another have symptoms and so on high fever, skin cyanosis, scant of breath, the course continue generally for 5~7 days, the morbidity pig take die generally as the extension turn over. In the disparity in age section's pig group has the varying degree occurrence, clinical manifestation basic similar. Split through the clinical symptoms and the pathology examine, I doubt that the sickness pigs mixed infection with PRRSV virus、PCV-2 virus 2 and E.suis. According to this,We decide to finish it in the southwest university of Rongchang campus animal disease fast diagnostic center.。

Key word: Porcine circovirus type 2 Eperythrozoon Mixed infection Integrated Control。

1 材料与方法

1．1Bio-Rad PCR仪、菲恰尔（GL-16A）离心机、JUNYI紫外灯（JY04S-3C）、苏静安泰超净工作台、Pipet.Lite微量可调移液器等。所有实验均在西南大学荣昌校区动物疾病快速诊断中心进行。

1．2猪圆环病毒（PCV）核酸扩增检测试剂盒、猪蓝耳病病毒（PRRSV）核酸扩增检测试剂盒均购自北京索奥生物技术有限公司。

2 临床症状

2．1病猪体温41～42℃，稽留热、精神沉郁、皮毛粗乱、心跳和呼吸加快、双侧性震颤，耳部、腹下、腹股沟、四肢出现不规则的紫斑，指压不褪色，有较浓的眼屎、眼结膜苍白、厌食或不食、呼吸困难、后躯无力、行走摇摆，病猪初期便秘，呈粪球状，小便深黄色，发病后期下痢和便秘交替出现。

3 病料采集

对濒死期的或刚刚死亡的猪进行仔细剖检，无菌操作下分别采集心血、肝、脾、肾等病料，供实验室检测用。

4 细菌学检查 。

4．1血液压片 取抗凝血或鲜血一滴，加3－4倍生理盐水稀释，盖上盖玻片，1000倍油镜下观察，可见大多数红细胞边缘呈锯齿状、星芒状或不规则状，并有微微颤动，即可诊断为附红细胞体感染阳性。判定标准:在油镜下检查20个视野，有25个以下附红细胞体的为感染+，有25－50个附红细胞体的为感染++，有50－70及70以上个附红细胞体的为感染+++。

4．2组织涂片 将无菌采取的肝、脾、肾及淋巴结涂片，革兰氏染色镜检。

5 PRRSV和PCV－2的PCR检测。

5．1核酸提取 病料RNA和DNA的提取分别用Trizol reagents(RNA提取液)和核酸提取液B（DNA）提取，按照说明书提供的方法进行。首先将血清稀释，取100ul释出血清加入500ul Trizol reagents（RNA提取液）充分震荡，混匀后室温放置5min。加入100ul氯仿，用力振荡15s，室温静置10min，4℃13，000 rpm离心5min。小心将上层水相转移到干净的离心管，加入等体积的异丙醇，充分混匀，13，000 rpm离心5min。弃上清，加入75％DEPC 乙醇，充分混匀，13，000 rpm离心5min，小心吸去大部分乙醇。将提取管口敞口在室温空气中干燥5min，待乙醇挥发干净，用20ul DEPC H2O溶解沉淀。

5．2逆转录 根据标本数取逆转录反应管，取2ul上述获得的RNA并加入7ul PRRSV－SM RT MIX引物进行逆转录。PRRSV 逆转录的反应体系为25ul，反应条件为：30℃5min，42℃45 min后98℃5min灭活转录酶。PCV－2 PCR检测则省去RNA提取与逆转录步骤，直接进行相应的扩增。

5．3扩增PCR PRRSV PCR扩增的反应体系为25ul。PRRSV PCR扩增反应条件为：94℃变性2min,93℃→2min，93℃ 30 s→58℃ 30s→72℃ 30s,共35个循环；PCV-2 PCR扩增反应条件为：93℃→2min，后93℃ 30s→58℃ 30s→72℃ 30s,共35个循环。

5．4电泳检测 取15ul PCR扩增产物直接用加样枪点样于2％琼脂糖凝胶胶孔中，以5 v/cm电压，1 x TAE中电泳20min后，紫外灯下观察结果。用DL2000 Marker作为参照。PRRSV和PCV－2的阳性扩增带分别为396bp和143bp。

6 结果

6．1剖检变化 濒死期病猪的典型表现为：病猪尸体消瘦，不同程度的贫血和黄疸，体表皮肤、粘膜黄染，血液稀薄呈水样；皮下水肿，呈黄白色；体表和肠系膜淋巴结充血、出血和水肿；淋巴结肿大4～5倍，切面外翻，尤以腹股沟淋巴结、肠系膜淋巴结、气管和支气管淋巴结明显，切面多汁呈黄色；肺部的病变主要是肿胀、间质增宽，有散在的橡皮样硬块；肝脏变性且有轻微黄疸、肿大，呈黄棕色；脾脏肿大；肾脏水肿，颜色呈灰白色，覆盖有小出血点。部分病猪心肌变软，心包积液。

6．2细菌学检查结果

6．2．1血液压片 1000倍油镜下观察，可见大多数红细胞边缘呈锯齿状、星芒状或不规则状,诊断为附红细胞体感染阳性，标记为：+++,结果见图2。

6．2．3组织涂片 革兰氏染色镜检，未见细菌。

6．3电泳检测结果：猪圆环病毒（PCV－2）为阳性；蓝耳病病毒（PRRSV）检测为阴性。结果见图1。

图1 PCV-2和PRRSV PCR检测结果 图2 血液压片油镜观察。

M：DL2000 Marker；1：PCV-2 PCR产物；2：PRRSV PCR产物。

7 防治措施

7．1该病目前尚无疫苗预防，改进饲养管理，对发病严重的病猪立即淘汰，发病轻微的猪进行隔离治疗。用2％ ～3％的烧碱对猪舍进行彻底消毒，保持猪舍饲养用具的清洁卫生；切断附红细胞体病的传播途径。给予全价饲料增加机体的抗病能力，减少不良应激的发生。改进饲养管理，减少应激是控制继发感染的关键。特别是仔猪断奶后的3～4周是预防该病最关键最有效的时期，要降低饲养密度，尽量减少猪群的应激因素，做好猪瘟、猪伪狂犬病、猪细小病毒病、猪蓝耳病疫苗的接种工作，做好猪群平时药物预防工作，母猪在产前1周和产后1周在饲料中可添加土霉素等抗菌药物，仔猪在断奶前1周至断奶后1个月在饲料中可添加强力霉素等抗菌药，同时也拌料一些有预防作用的中药，如清温败毒散进行预防，以减少该病在猪群中流行。

7．2猪圆环病毒是猪的免疫抑制性疾病，引起和继发感染的疾病已成为世界养猪业的大敌。目前仍无有效防治措施，采取的方法是控制继发感染，特别要加强断奶仔猪的营养和管理，采取药物预防，降低猪群死亡率，减少经济损失。

7．3对症治疗

7．3．1治疗圆环病毒病目前尚无特效的治疗药物，应早发现，早确诊，早治疗。对发病不严重的猪只，选用新型的抗病毒剂如干扰素、白细胞介导素、免疫球蛋白、转移因子等进行治疗；同时采用黄芪多糖注射液并配合Vb1、Vb12、Vc肌肉注射，增强猪自身免疫力；另外，也要采用抗菌药物，控制继发感染，如氟苯尼考、阿米卡星、盐酸林可壮观霉素等配合治疗，尽量少用磺胺类药物和地塞米松进行治疗。

7．3．2目前对猪附红细胞体病疗效较好的药物有长效土霉素，四环素，贝尼尔等，按说明剂量肌注24h后，可见临床症状改善，连用3～5d。另外，对阳性猪群的贫血猪，可应用铁制剂(右旋糖酐铁)、土霉素、Vb12、VC等对症治疗。

8 小结与讨论

8．1此次疫病根据流行病学、临床症状和剖检变化及实验室诊断，最后确诊为猪圆环病毒2型与附红细胞体混合感染。

8．2猪圆环病毒病是由猪圆环病毒感染所引起的以免疫抑制为特征的一类病毒性传染病。PCV-2感染新生仔猪及断奶仔猪，使仔猪发生先天性震颤和断奶后全身消耗性综合征，是一种慢性消耗性疾病，导致断奶仔猪生长缓慢、发育障碍、机体免疫力下降、易继发其他疾病(附红细胞体病、弓形体病、猪瘟等)而加重病情。要抓住关键时期，减少应激，仔猪断奶后3～4周，是预防圆环病毒的最关键时期，最有效的方法和措施是尽可能减少断奶仔猪的应激。经分析，猪长途贩运，使动物机体产生一定的应激反应，加上抵抗力下降，特别是感染附红体和弓形体病后，进一步降低机体的免疫力，当遇到传染性疾病流行时，特别是遇到猪圆环病毒感染侵袭，就会造成病猪死亡率增高。

8．3猪附红细胞体病是由猪附红细胞体引起猪的一种血液病，蚊虫是重要的传播媒介，尤其在多雨季节蚊虫易孳生，加上猪群感染圆环病毒，机体抵抗力下降，而发生猪附红细胞体病。防治该病应消灭蚊虫，切断传播途径，加强器械的消毒，提高管理水平。从现在的季节来看，正是春季，气温时高时低，忽热忽冷，春季又是病菌活动频繁季节，所以加快了疫病流行。另外，老鼠可传播多种疾病，若鼠害控制不力，则易引起疾病的发生，因此，必须控制鼠害。

8．4为了减少疾病的发生，规模化养猪场必须坚持自繁自养，做到全进全出，加强平时消毒工作，这样尽可能地避免或减少疫病的传入和流行。

8．5附红细胞体病的死亡率很低，但与猪圆环病毒混合感染后，死亡率就会显著上升，可达60％以上，这也是造成病猪死亡率增高的重要原因。采用综合措施后，该猪场的病情基本稳定，得到了有效控制，到2月下旬逐渐恢复正常。

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[13]Messick J B，Cooper S K，Huntley M．Development and evalu—ation of polymerase chain reaction assay using the 16S rRNA gene for detection of Eperythrozoon suis infection [J]．J Vet Diagn Invest，1999，11(3)：229—236．。

[14] 王宏，金扩世，马呜啸. 猪蓝耳病和圆环病混合感染的诊断与防治[J].吉林畜牧兽医，2007,28(10): 32-33.。

[15] 李继超,徐克发,冉宇生,向绍平. 猪蓝耳病的诊治[J]. 畜牧市场,2008, (8)：65-66.。

[16] 王芳,鄢珣,韩庆彦. 猪2型圆环病毒PCR检测方法的建立及应用[J]. 甘肃畜牧兽医, 2008,38(4): 13-16.。

[18] 阎金龙,张志勇,易春玲. 圆环病毒与附红细胞体混合感染的诊治[J]. 吉林畜牧兽医, 2008, 29(4): 24-25.。

[19] 周凤山,魏桂兰,季燚,徐增强. 猪附红细胞体病并发圆环病毒病的诊治[J].畜禽业,2008,(3): 85-86.。

[20] 何晓剑,韩建珍. 猪圆环病毒的防制[J]. 河南畜牧兽医,2008,29(10): 44-45.。

[21] 董信田,肖剑,金俊杰,金大春,王跃川. 猪圆环病毒2型PCR检测方法的建立[J]. 畜禽业,2008,(90)：22-24.。

[22] 王林,王睿, 郭艳杰. 猪圆环病毒与附红细胞体混合感染的诊治[J]. 四川畜牧医,2005,32(4): 44-45.。

[23] PCR detection of porcine circovirus type 2 (PCV2) DNA in blood, tonsillar and faecal swabs from experimentally infected pigs A. Caprioli[a]; F. McNeilly[b]; I. McNair[b]; P. Lagan-Tregaskis[b]; J.Ellis[c];S.Krakowka[d];J.McKillen[b]; F. Ostanello[a]; G. Allan[b] Research in Veterinary Science, Volume 81, Issue 2, October 2006, Pages 287-292．。

[24]The involvement of Fas/FasL interaction in porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus co-inoculation-associated lymphocyte apoptosis in vitro Hui-Wen Chang[a]; Chian-Ren Jeng[a]; Chun-Ming Lin[a]; Jiuan Judy Liu[a][b]; Chih-Cheng Chang[c]; Yi-Chieh Tsai[a]; Mi-Yuan Chia[a]; Victor Fei Pang[a][d]&Email:pang@ntu.edu.tw ．Veterinary Microbiology, Volume 122, Issue 1-2, 16 May 2007, Pages 72-82．。

建议看看汪家政 的《 蛋白质技术手册》，上面写得比较详细。

AMERSHAM的PROTOCOL:。

Under native PAGE conditions, polypeptides retain their higher-order structure and often retain enzymatic activity and interaction with other polypeptides. The migration of proteins depends on many factors, including size, shape, and native charge. The resolution of nondenaturing electrophoresis is generally not as high as that of SDS-PAGE, but the technique is useful when the native structure or enzymatic activity of a protein must be assayed following electrophoresis. One straightforward approach to native electrophoresis is to omit the SDS and reducing agent (DTT) from the standard Laemmli SDS protocol. Prepare the gels as described above, but do not put SDS or DTT in the sample buffer, do not heat the samples, and prepare the gel and tank buffer solutions without SDS.。

Nondenaturing Polyacrylamide Gel Electrophoresis of Proteins。

1. Introduction。

SDS-PAGE (Section 2.2) is probably the most commonly used gel electrophoretic system for analyzing proteins.。

However, it should be stressed that this method separates denatured protein. Sometimes one needs to analyze native,nondenatured proteins, particularly if wanting to identify a protein in the gel by its biological activity (for example, enzyme activity, receptor binding, antibody binding, and so on). On such occasions it is necessary to use a nondenaturing system such as described in this section. For example, when purifying an enzyme, a single major band on a gel would suggest a pure enzyme. However this band could still be a contaminant; the enzyme could be present as a weaker (even nonstaining) band on the same gel. Only by showing that the major band had enzyme activity would you be convinced that this band corresponded to your enzyme. The method described here is based on the gel system first described by Davis ( 1). To enhance resolution a stacking gel can be included (see Section 2.2 for the theory behind the stacking gel system).。

2. Materials

1. Stock acrylamide solution: 30 g acrylamide, 0.8 g bis-acrylamide. Make up to 100 mL in distilled water and filter. Stable at 4°C for months (see Note 1). Care: Acrylamide Monomer Is a Neurotoxin. Take care in handling acrylamide (wear gloves) and avoid breathing in acrylamide dust when weighing out.。

2. Separating gel buffer: 1.5M Tris-HCl, pH 8.8.。

3. Stacking gel buffer: 0.5M Tris-HCl, pH 6.8.。

4. 10% Ammonium persulfate in water.。

5. N,N,N',N'-tetramethylethylenediamine (TEMED).。

6. Sample buffer (5X). Mix the following:。

a. 15.5 mL of 1M Tris-HCl pH 6.8;。

b. 2.5 mL of a 1% solution of bromophenol blue;。

c. 7 mL of water; and。

d. 25 mL of glycerol.。

Solid samples can be dissolved directly in 1X sample buffer. Samples already in solution should be diluted。

accordingly with 5X sample buffer to give a solution that is 1X sample buffer. Do not use protein solutions that are in a strong buffer that is not near to pH 6.8 as it is important that the sample is at the correct pH. For these samples it will be necessary to dialyze against 1X sample buffer.。

7. Electrophoresis buffer: Dissolve 3.0 g of Tris base and 14.4 g of glycine in water and adjust the volume to 1 L. The final pH should be 8.3.。

8. Protein stain: 0.25 g Coomassie brilliant blue R250 (or PAGE blue 83), 125 mL methanol, 25 mL glacial acetic。

acid, and 100 mL water. Dissolve the dye in the methanol component first, then add the acid and water. Dye。

solubility is a problem if a different order is used. Filter the solution if you are concerned about dye solubility. For。

best results do not reuse the stain.。

9. Destaining solution: 100 mL methanol, 100 mL glacial acetic acid, and 800 mL water.。

10. A microsyringe for loading samples.。

3. Method

1. Set up the gel cassette.。

2. To prepare the separating gel (see Note 2) mix the following in a Buchner flask: 7.5 mL stock acrylamide solution, 7.5 mL separating gel buffer, 14.85 mL water, and 150 L 10% ammonium persulfate. "Degas" this solution under vacuum for about 30 s. This degassing step is necessary to remove dissolved air from the solution, since oxygen can inhibit the polymerization step. Also, if the solution has not been degassed to some extent, bubbles can form in the gel during polymerization, which will ruin the gel. Bubble formation is more of a。

problem in the higher percentage gels where more heat is liberated during polymerization.。

3. Add 15 L of TEMED and gently swirl the flask to ensure even mixing. The addition of TEMED will initiate the polymerization reaction, and although it will take about 20 min for the gel to set, this time can vary depending on room temperature, so it is advisable to work fairly quickly at this stage.。

4. Using a Pasteur (or larger) pipet, transfer the separating gel mixture to the gel cassette by running the solution。

carefully down one edge between the glass plates. Continue adding this solution until it reaches a position 1 cm from the bottom of the sample loading comb.。

5. To ensure that the gel sets with a smooth surface, very carefully run distilled water down one edge into the cassette using a Pasteur pipet. Because of the great difference in density between the water and the gel solution, the water will spread across the surface of the gel without serious mixing. Continue adding water until a layer about 2 mm exists on top of the gel solution.。

6. The gel can now be left to set. When set, a very clear refractive index change can be seen between the polymerized gel and overlaying water.。

7. While the separating gel is setting, prepare the following stacking gel solution. Mix the following quantities in a。

Buchner flask: 1.5 mL stock acrylamide solution, 3.0 mL stacking gel buffer, 7.4 mL water, and 100 L 10%。

ammonium persulfate. Degas this solution as before.。

8. When the separating gel has set, pour off the overlaying water. Add 15 L of TEMED to the stacking gel solution and use some (~2 mL) of this solution to wash the surface of the polymerized gel. Discard this wash, then add the stacking gel solution to the gel cassette until the solution reaches the cutaway edge of the gel plate. Place the well-forming comb into this solution and leave to set. This will take about 30 min. Refractive index changes around the comb indicate that the gel has set. It is useful at this stage to mark the positions of the bottoms of the wells on the glass plates with a marker pen.。

9. Carefully remove the comb from the stacking gel, remove any spacer from the bottom of the gel cassette, and。

assemble the cassette in the electrophoresis tank. Fill the top reservoir with electrophoresis buffer ensuring that the buffer fully fills the sample loading wells, and look for any leaks from the top tank. If there are no leaks, fill the bottom tank with electrophoresis buffer, then tilt the apparatus to dispel any bubbles caught under the gel.。

10. Samples can now be loaded onto the gel. Place the syringe needle through the buffer and locate it just above the bottom of the well. Slowly deliver the sample (~5-20 L) into the well. The dense sample solvent ensures that the sample settles to the bottom of the loading well. Continue in this way to fill all the wells with unknowns or。

standards, and record the samples loaded.。

11. The power pack is now connected to the apparatus and a current of 20-25 mA passed through the gel (constant。

current) (see Note 3). Ensure that the electrodes are arranged so that the proteins are running to the anode (see Note 4). In the first few minutes the samples will be seen to concentrate as a sharp band as it moves through the stacking gel. (It is actually the bromophenol blue that one is observing, not the protein but, of course, the protein is stacking in the same way.) Continue electrophoresis until the bromophenol blue reaches the bottom of the gel. This will usually take about 3 h. Electrophoresis can now be stopped and the gel removed from the cassette. Remove the stacking gel and immerse the separating gel in stain solution, or proceed to step 13 if you wish to detect enzyme activity (see Notes 5 and 6).。

12. Staining should be carried out, with shaking, for a minimum of 2 h and preferably overnight. When the stain is。

replaced with destain, stronger bands will be immediately apparent and weaker bands will appear as the gel destains.Destaining can be speeded up by using a foam bung, such as those used in microbiological flasks. Place the bung in the destain and squeeze it a few times to expel air bubbles and ensure the bung is fully wetted. The bung rapidly absorbs dye, thus speeding up the destaining process.。

13. If proteins are to be detected by their biological activity, duplicate samples should be run. One set of samples should be stained for protein and the other set for activity. Most commonly one would be looking for enzyme activity in the gel. This is achieved by washing the gel in an appropriate enzyme substrate solution that results in a colored product appearing in the gel at the site of the enzyme activity (see Note 7).。

4. Notes

1. The stock acrylamide used here is the same as used for SDS gels (see Section 2.2) and may already be availabe in your laboratory.。

2. The system described here is for a 7.5% acrylamide gel, which was originally described for the separation of serum proteins (1). Since separation in this system depends on both the native charge on the protein and separation according to size owing to frictional drag as the proteins move through the gel, it is not possible to predict the electrophoretic behavior of a given protein the way that one can on an SDS gel, where separation is based on size alone. A 7.5% gel is a good starting point for unknown proteins. Proteins of mol wt >100,000 should be separated in 3-5% gels. Gels in the range 5-10% will separate proteins in the range 20,000-150,000, and 10-15% gels will separate proteins in the range 10,000-80,000. The separation of smaller polypeptides is described in Section 2.7. To alter the acrylamide concentration, adjust the volume of stock acrylamide solution in Subheading 3., step 2 accordingly, and increase/decrease the water component to allow for the change in volume. For example, to make a 5% gel change the stock acrylamide to 5 mL and increase the water to 17.35 mL. The final volume is still 30 mL, so 5 mL of the 30% stock acrylamide solution has been diluted in 30 mL to give a 5% acrylamide solution.。

3. Because one is separating native proteins, it is important that the gel does not heat up too much, since this could。

denature the protein in the gel. It is advisable therefore to run the gel in the cold room, or to circulate the buffer。

through a cooling coil in ice. (Many gel apparatus are designed such that the electrode buffer cools the gel plates.) If heating is thought to be a problem it is also worthwhile to try running the gel at a lower current for a longer time.。

4. This separating gel system is run at pH 8.8. At this pH most proteins will have a negative charge and will run to the anode. However, it must be noted that any basic proteins will migrate in the opposite direction and will be lost from the gel. Basic proteins are best analyzed under acid conditions, as described in Sections 2.5 and 2.6.。

5. It is important to note that concentration in the stacking gel may cause aggregation and precipitation of proteins.。

Also, the pH of the stacking gel (pH 6.8) may affect the activity of the protein of interest. If this is thought to be a。

problem (e.g., the protein cannot be detected on the gel), prepare the gel without a stacking gel. Resolution of。

proteins will not be quite so good, but will be sufficient for most uses.。

6. If the buffer system described here is unsuitable (e.g., the protein of interest does not electrophorese into the gel。

because it has the incorrect charge, or precipitates in the buffer, or the buffer is incompatible with your detection。

system) then one can try different buffer systems (without a stacking gel). A comprehensive list of alternative buffer systems has been published (2).。

7. The most convenient substrates for detecting enzymes in gels are small molecules that freely diffuse into the gel and are converted by the enzyme to a colored or fluorescent product within the gel. However, for many enzymes such convenient substrates do not exist, and it is necessary to design a linked assay where one includes an enzyme。

together with the substrate such that the products of the enzymatic reaction of interest is converted to a detectable。

product by the enzyme included with the substrate. Such linked assays may require the use of up to two or three。

enzymes and substrates to produce a detectable product. In these cases the product is usually formed on the urface。

of the gel because the coupling enzymes cannot easily diffuse into the gel. In this case the zymogram technique is。

used where the substrate mix is added to a cooled (but not solidified) solution of agarose (1%) in the appropriate。

buffer. This is quickly poured over the solid gel where it quickly sets on the gel. The product of the enzyme assay is therefore formed at the gel-gel interface and does not get washed away. A number of review articles have been。

published which described methods for detecting enzymes in gels (3-7). A very useful list also appears as an。

appendix in ref. 8.。

References

1. Davis, B. J. (1964) Disc electrophoresis IImethod and application to human serum proteins. Ann. NY Acad. Sci. 121, 404-427.。

2. Andrews, A. T. (1986) Electrophoresis: Theory, Techniques, and Biochem-ical and Clinical Applications.。

Clarendon, Oxford, UK.。

3. Shaw, C. R. and Prasad, R. (1970) Gel electrophoresis of enzymesa compilation of recipes. Biochem. Genet. 4, 297-320.。

4. Shaw, C. R. and Koen, A. L. (1968) Starch gel zone electrophoresis of enzymes, in Chromatographic and。

Electrophoretic Techniques, vol. 2 (Smith, I., ed.), Heinemann, London, pp. 332-359.。

5. Harris, H. and Hopkinson, D. A. (eds.) (1976) Handbook of Enzyme Electrophoresis in Human Genetics.。

North-Holland, Amsterdam.。

6. Gabriel, O. (1971) Locating enymes on gels, in Methods in Enzymology, vol. 22 (Colowick, S. P. and Kaplan, N. O., eds.), Academic, New York, p. 578.。

7. Gabriel, O. and Gersten, D. M. (1992) Staining for enzymatic activity after gel electrophoresis. I. Analyt. Biochem. 203, 1-21.。

8. Hames, B. D. and Rickwood, D. (1990) Gel Electrophoresis of Proteins, 2nd ed., IRL, Oxford and Washington.。

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