TCA-丙酮沉淀法浓缩蛋白
真菌蛋白提取方法
真菌蛋白提取方法一TCA/丙酮法1 液氮将菌体研磨为粉末；2 向匀浆中加入10倍体积的丙酮（含13.3% (w/v) TCA和0.093% β巯基乙醇），过夜沉降；3 4 度20,000g 离心15 min；4 移处上清，用冰冷的含0.07% (v/v) β－巯基乙醇的丙酮清洗沉淀两次；5 离心后，沉淀干燥至丙酮挥发完全。
二酚抽提法1 在液氮中将 1g 菌体研磨为粉末2 加入 6 ml 水饱和酚（buffer-saturated phenol）和 2 ml提取液（20mMTris-Cl, pH 7.5, 0.5% Triton X-100 (v/v), 0.5M EDTA, pH 7.5, pH 8.0,0.07% β－巯基乙醇, protease inhibitors）。
3 在4度混合30分钟。
4 12000 rmp 离心1min，使两相分开。
5 弃沉淀，吸取上层的苯酚相，加入5倍体积的冷丙酮，-20度沉降2h。
6 12000 rmp，4度，离心15分钟沉淀蛋白，弃去上清。
7 沉淀干燥至丙酮挥发完全。
三提取液抽提丙酮沉淀法1 预先将研钵置于－20℃的冰箱内冷冻。
2 取液氮冻存的菌体，放入预冷的研钵内研磨至粉末状。
3 向匀浆中加入2倍体积的抽提液（20mM Tris-Cl, pH 7.5, 0.5% Triton X-100(v/v), 0.5M EDTA, pH 7.5, pH 8.0, 0.07% β－巯基乙醇, protease inhibitors）。
4 混匀后4度震荡30分钟，以便蛋白溶解。
5 12000 rmp，4度，离心15分钟，吸取上清于另一EP管中。
7 向上清液中加入5倍体积的预冷丙酮（含有0.07％β－巯基乙醇），混匀，－20℃，2小时沉淀蛋白质。
8 12000 rmp，4度，离心15分钟沉淀蛋白。
弃去上清，沉淀用乙醇洗三次，自然干燥蛋白沉淀。
四甲醇氯仿水沉淀法Chloroform /Methanol/Water Precipitation1 预先将研钵置于－20℃的冰箱内冷冻。
TCA蛋白沉淀方法
100% (w/v)三氯乙酸的配制方法：500g三熬乙酸用227ml水来溶無，所得溶液即100%三氯乙酸溶液。
避光,4度保(preparation of 100% TCA: 454ml H2O/kgTCA. Maintain in dark bold cat 4oC.Bc careful, use gloves!!!).培养基上清直接电泳跑出来的条带经常很难看，可以TCA沉淀浓缩后跑电泳，一般表达量大于可以看到明显条带，这是我用的TCA沉淀方法，效果很好：1 •菌液1000(也，离心5分钟，收集表达上清。
2•取500-10(X)ul ±清于EP管中，加入1/9体积的100%TCA,颠倒1()次混匀。
3•样品置于冰浴中大于0.5小时，过夜效果更好。
4.15000g,离心10-20分钟,可见有棕黑色沉淀，倒掉上清，将EP管倒扣在吸水纸上轻轻控几下，除去残余在管口的液体。
5.将EP管倒置于吸水纸长，37度烘箱10-20分钟，待管底无明显液体残留，如杲管壁还残留有液体，可以吸水纸吸掉。
可以改成室温或用电吹风，关犍是除去管底和管壁残余液体。
6.15OOOg,离心10-20分钟，用20ul枪头尽量吸去管底残余的液体，此步骤要快，不然沉淀容易散开，降低蛋白回收率，一般最多几ul或者没有，注意不要吸到沉淀。
7.EP管倒置于吸水纸长，37度烘箱5分钟，确认管壁和管底没有液体残留。
&加入20-50ul Loading buffer, 95度加热lOnim, —般沉淀会自动溶解，如果不溶，用手指轻弹管壁或用2()ul枪头轻轻吸打，注意整个操作尽量不要碰到管壁, 因为管壁可能沾有残余TCA。
如果蓝色的Loading buffer不变成黄色，说明残余TCA吸弃了干净，如果变黄，一般不影响电泳。
此方法连丙酮洗这一步都省了，而且不影响电泳效杲。
或者第5步和第6步改为丙酮洗:5•加入2()0u】冰冷的丙酮，用手指轻弹EP管，洗去管底和管壁残余的TCA。
植物组织蛋白提取方法
植物蛋白质提取方法总汇一、植物组织蛋白质提取方法1、根据样品重量（1g 样品加入3.5ml 提取液，可根据材料不同适当加入），准备提取液放在冰上。
2、把样品放在研钵中用液氮研磨，研磨后加入提取液中在冰上静置（3-4 小时）。
3、用离心机离心8000rpm40min4 C或11100rpm20min4 °C4、提取上清液，样品制备完成。
蛋白质提取液：300ml1 、1Mtris-HCl （PH8）45ml2、甘油（Glycerol ）75ml3、聚乙烯吡咯烷酮（Polyvinylpolypyrrordone ）6g这种方法针对SDS-PAGE垂直板电泳！二、植物组织蛋白质提取方法氯醋酸—丙酮沉淀法1 、在液氮中研磨叶片2、加入样品体积3倍的提取液在-20 C的条件下过夜，然后离心（4 C 8000rpm以上1 小时）弃上清。
3、加入等体积的冰浴丙酮（含0.07%的3 -巯基乙醇），摇匀后离心（4C 8000rpm 以上1 小时），然后真空干燥沉淀，备用。
4、上样前加入裂解液，室温放置30 分钟，使蛋白充分溶于裂解液中，然后离心（15C 8000rpm以上1小时或更长时间以没有沉淀为标准），可临时保存在 4 C待用。
5、用Brandford法定量蛋白，然后可分装放入-80 C备用。
药品：提取液：含10%TCA和0.07%的3 -巯基乙醇的丙酮。
裂解液：2.7g尿素0.2gCHAPS 溶于3ml灭菌的去离子水中（终体积为5ml），使用前再加入1M的DTT65ul/ml。
这种方法针对双向电泳，杂质少，离子浓度小的特点！当然单向电泳也同样适用，只是电泳的条带会减少！三、组织：肠黏膜目的：WESTERN BLO检测凋亡相关蛋白的表达应用TRIPURE提取蛋白质步骤：含蛋白质上清液中加入异丙醇：（1.5ml每ImITRIPURE用量）倒转混匀，置室温10min离心：12000 g，10min，4 度，弃上清加入0.3M盐酸胍/95 %乙醇：（2ml每ImITRIPURE用量）振荡，置室温20min离心：7500g ，5 min ，4 度，弃上清重复0.3M 盐酸胍/95 %乙醇步2 次沉淀中加入100%乙醇2ml充分振荡混匀，置室温20 min离心：7500g ，5min，4 度，弃上清吹干沉淀1 % SDS溶解沉淀离心：10000g，10min ，4 度取上清-20 度保存（或可直接用于WESTERN BLO）T存在的问题：加入1%SDS后沉淀不溶解，还是很大的一块，4度离心后又多了白色沉定，SDS结晶？测浓度，含量才1mg/ml左右。
TCA-丙酮沉淀法蛋白提取
TCA-丙酮沉淀法蛋白提取仪器：Eppendorf 冷冻离心机(Eppendorf)、组织匀浆器、超声破碎仪主要溶液配置：1.10mL SDS裂解液1%DTT，2% SDS，10%甘油，50mM Tris-Hcl(pH6.8)。
配置：称取0.1g DTT，0.2g SDS，加入1mL甘油和1mL的0.5MTris-Hcl(pH6.8)，然后定容至10mL。
2.10mL Triton样品溶解液浓度：150mM NaCl，1％Triton-X-100，50mM Tris-HCl(pH8.0)配置：称取0.087gNaCl、加入0.1mL Triton-X-100、500μL 浓度为1M的Tris-HCl (pH8.0) 加入约9.4mL水，混合溶解均匀。
保存：4度保存。
3.1mL尿素样品溶解液（现配现用）浓度：9M 尿素，4％CHAPS，1% IPGBuffer，1% DTT,配置：称取0.54g尿素、加入0.04g CHAPS、再加入0.56mL双蒸水，溶解后分装-20度冷冻保存，使用前融解后按0.98mL加入0.01g DTT（DTT过早加入会失效）和10μL Buffer的比例混合溶解均匀，最后再加入痕量溴酚蓝。
保存：-20度保存。
4.RIPA裂解液：浓度：50 mmol/L Tris-HCl (pH 8.0)，150 mmol/L NaCl，0.2 g/L叠氮钠，1 g/LSDS 100 mg/L Aprotin，10 g/L NP-40，5 g/L去氧胆酸钠，100 mg/LPMSF。
(RIPA裂解液有强、中和弱三种类型，其试配置略有差异)配置：称取：0.07882g Tris-HCl，0.08775g NaCl，0.002g叠氮钠，0.01gSDS，0.001g Aprotin，0.1g NP-40，0.05g去氧胆酸钠，0.001g PMSF。
注：以上列举了一些常见的裂解液，除此之外还有多种类型的裂解液，需要研究者根据自己研究的材料和实验目的选择合适的裂解液提取蛋白。
烟草叶片蛋白3种提取方法的比较研究
烟草叶片蛋白3种提取方法的比较研究摘要:使用酚/SDS法､Tris-HCl法及TCA/丙酮沉淀法提取烟草(Nicotiana tabacum)叶片蛋白,从终产物形式､颜色､提取时间､蛋白产量､SDS-PAGE电泳图谱等方面对3种方法进行比较,对提取的烟草叶片蛋白进行Western Blotting检测来评价3种方法提取蛋白的效果｡结果表明,与2种常用方法相比,酚/SDS法具有快速､操作方便等特点,提取的蛋白纯度高､种类丰富,可用于Western Blotting检测,是一种合适的提取植物组织中蛋白的方法｡关键词:烟草(Nicotiana tabacum)叶片;蛋白;Tris-HCl法;TCA/丙酮沉淀法;酚/SDS法Comparison Study on Three Methods for Tobacco Leaf Protein Extraction Abstract: Protein was extracted from tobacco(Nicotiana tabacum) leaves by three methods, phenol/SDS method, Tris-HCl method and TCA/acetone precipitation method. The extracting efficiency of the three methods was evaluated through the color and form of product, extraction time, protein yield and SDS-PAGE. Furthermore, the extracted protein was detected by Western Blotting. The results showed that compared with the other two methods, phenol/SDS method was faster and more convenient, and the protein extracted was with high quality, rich in protein species, and suitable for Western Blotting, thus was an appropriate method for extracting protein from plant tissue.Key words: tobacco(Nicotiana tabacum) leaf; protein; Tris-HCl extraction method; TCA/acetone precipitation; phenol/SDS extraction method蛋白质组学(Proteomics)是随着基因组学发展而发展起来的､在整体水平上研究细胞内蛋白质的组成及其活动规律的学科｡由于其能够阐明基因表达产物——蛋白在生物体内的相对含量､修饰化信息,蛋白质与其他生物大分子的相互作用等诸多内容而日益成为现代生物学的研究热点｡在蛋白质组研究中,蛋白样品制备是分析的起始和基础,蛋白提取质量和效果对后续的研究分析有重要影响[1,2]｡植物组织含有较厚的细胞壁,给组织中蛋白质的提取增加了一定的难度｡目前常用的植物蛋白的提取方法有两种,一种是普通Tris-HCl提取法,该方法通过选择适当的提取缓冲液pH,将植物中的可溶性蛋白尽可能地溶解;另一种是TCA/丙酮沉淀法,该方法中TCA作为蛋白质变性剂,能有效抑制丝氨酸蛋白酶和巯基蛋白酶的活性,减少蛋白损失,因此得到较为广泛的使用[3-6]｡但以上两种方法都不能有效地去除产物中的非蛋白物质,会对后续的研究产生不利的影响｡而酚/SDS蛋白提取法中,利用酚在SDS这种阴离子型表面活性剂的存在条件下能充分溶解蛋白的特点,可以取得在较短的时间内充分溶解植物组织中蛋白的效果,得到的蛋白纯度更高[7,8]｡目前该方法在国内使用得较少,特别是在烟草样品中尚未见报道｡本研究同时使用酚/SDS法､Tris-HCl法及TCA/丙酮沉淀法3种方法从烟草叶片中提取蛋白质,从终产物形式､颜色､提取时间､蛋白产量､SDS-PAGE电泳结果等方面进行比较,并使用糖基转移酶抗体对提取的烟草叶片蛋白进行免疫学检测,以评价3种方法提取蛋白的效果,为植物蛋白的提取和蛋白组学研究提供参考｡1 材料与方法1.1 材料和试剂烟草叶片(W38)来自中南民族大学生命科学学院国家民委生物技术重点实验室｡主要试剂:NC膜购自Whatman公司;ECL Western Blot System购自碧云天公司;Tris､三氯乙酸等化学试剂为国产分析纯;糖基转移酶多克隆抗体由北京华大蛋白技术有限公司制备｡1.2 蛋白提取方法1.2.1 Tris-HCl提取法准确称取0.5 g叶片,剪碎后加入0.25 mL Tris-HCl溶液冰浴研磨,再加入0.75 mL提取液(7 mol/L尿素､2 mol/L硫脲､0.4% CHAPS､10 mmol/L DTT),研磨至匀浆后,转移至1.5 mL离心管中,10 000 r/min离心10 min,上清液为所需的总蛋白｡1.2.2 TCA/丙酮沉淀法取0.5 g叶片,用液氮研磨充分,加入5 mL预冷的TCA 提取液(含质量分数10%的TCA､20 mmol/L DTT､1 mmol/L PMSF的丙酮溶液)充分混合后,-20 ℃放置1 h;13 000 r/min､4 ℃离心15 min,取沉淀;再加入5 mL预冷的样品洗涤液(20 mmol/L DTT､1 mmol/L PMSF的丙酮溶液),充分混合后,-20 ℃放置1 h;13 000 r/min､4 ℃离心15 min,取沉淀;用密封膜封口,用针在膜上扎几个小洞,低温冷冻干燥｡置于-80 ℃保存或直接进行蛋白电泳｡1.2.3 酚/SDS蛋白提取法取0.5 g烟草样品,液氮中研磨至粉末,快速转移至7 mL试管,加入5 mL预冷的质量分数为10%的TCA丙酮溶液,振荡混合均匀后,12 000 r/min､4 ℃离心3 min,弃上清;加入5 mL含有0.1 mol/L乙酸铵的体积分数为80%的乙醇溶液,振荡混合后12 000 r/min､4 ℃离心3 min,弃上清;再加入5 mL体积分数80%的丙酮,涡旋振荡直至沉淀充分分散,12 000 r/min､4 ℃离心3 min,弃上清,室温干燥10 min以除去剩余的丙酮,加入2.5 mL苯酚/SDS溶液,振荡混匀后,放置5 min,12 000 r/min､4 ℃离心3 min;转移上层苯酚相(约1~2 mL)至新的7 mL 管中,加入含有0.1 mol/L乙酸铵的乙醇溶液,置-20 ℃中10 min或过夜,12 000 r/min､4 ℃离心5 min,小心弃上清｡用无水乙醇洗沉淀,再用体积分数80%的丙酮洗沉淀｡最后让蛋白自然干燥,或根据实验需要用缓冲液溶解蛋白,保存在-80 ℃冰箱,或直接进行蛋白电泳｡1.3 检测方法考马斯亮兰G-250法测蛋白含量[9];SDS-PAGE电泳检测,按照文献[10,11]所述方法进行;Western Blotting检测:取发育状况接近的烟草叶片,分别用3种方法提取总蛋白,进行SDS-PAGE电泳,然后通过蛋白转膜､丽春红染色检测､封闭､标记一抗､洗脱一抗､标记二抗和杂交､再洗脱､化学发光法显色检测结果等步骤进行分析[12,13]｡2 结果与分析2.1 3种方法提取的蛋白质质量和产量比较对3种提取方法得到的烟草叶片蛋白形式､颜色､提取时间及蛋白产量进行比较(表1),可以看出,Tris-HCl提取法所得产物为绿色,这是由色素等杂质成分未能被完全去除而导致｡该方法虽提取速度快､产量高,但蛋白质量不高,所含杂质较多｡TCA/丙酮沉淀法用液氮充分研磨烟草叶片,同时加入蛋白酶抑制剂PMSF,降低了蛋白降解损失,故蛋白产量最高｡但是多次洗脱后样品颜色仍为淡黄色,这可能是因为烟草叶片中的一些物质能与蛋白共沉淀,导致终产物含有杂质｡此外,此方法操作耗时较长,会导致蛋白样品的损失和降解｡使用酚/SDS法提取蛋白,酚能够有效溶解蛋白,并在SDS存在下,其溶解效果更好,可溶解的蛋白更多,且该方法能有效去除非蛋白物质,非蛋白类成分较其他两种方法都少,获得的产物为白色｡虽然该方法的蛋白产量较TCA/丙酮沉淀法低,提取所需时间长于Tris-HCl法,但蛋白较纯｡2.2 SDS-PAGE检测结果使用常规的SDS-PAGE对3种方法获得的烟草蛋白样品进行比较分析,在蛋白上样量为20 μg时,Tris-HCl法提取的蛋白中检测到10个条带,TCA/丙酮沉淀法提取的蛋白电泳检测出14条条带,酚/SDS法提取的蛋白有12条条带(图1)｡Tris-HCl 法提取的蛋白条带较少,说明只提取到了部分蛋白,可能是该方法采用冰浴研磨,细胞破碎不够充分,只能得到植物组织中的部分可溶性蛋白｡当蛋白上样量为40 μg时,酚/SDS法所得的蛋白条带最多(图2),说明该方法提取的蛋白种类最多,提取效果比较好｡2.3 Western Blotting检测结果使用Western Blotting,以烟草糖基转移酶抗体为探针检测3种方法获得的蛋白,结果显示普通Tris-HCl法和TCA/丙酮法提取的蛋白均未出现任何杂交信号,只有酚/SDS法提取的蛋白出现杂交信号(图3),这表明酚/SDS法提取的蛋白用于Western Blotting分析能得到较好的效果｡3 结论3.1 蛋白提取质量在蛋白提取中,杂质的去除是一个主要问题[2],本研究中酚/SDS法提取的蛋白颜色最浅,所含杂质少,有利于后续的蛋白质组分析｡另外,该方法步骤简单,提取时间较短,能在4~5 h内完成,具有操作方便､快速的特点｡其他两种方法提取的蛋白质均含有较多杂质,不利于后续实验的进行｡3.2 电泳和Western Blotting检测对3种方法提取的蛋白进行电泳检测,Tris-HCl法只提取到了部分蛋白｡TCA/丙酮沉淀法所得蛋白条带虽多,但是该方法需要的时间长,容易产生蛋白样品的降解,从而导致蛋白电泳结果不稳定｡而酚/SDS蛋白提取法所得条带较多,重复性好,能够得到良好的一维电泳图谱｡此外,本研究使用Western Blotting对烟草叶片蛋白中的糖基转移酶进行了检测｡结果显示酚/SDS法提取的烟草叶片蛋白表现良好,可用于后续分析｡酚/SDS法提取植物蛋白,可获得高质量和较高产量的目标产物,为有效提取植物叶片蛋白提供了有力的依据,也为后续的蛋白分析实验打下了良好的基础｡参考文献:[1] WANG W, VIGNANI R, SCALI M, et al. A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis[J]. Electrophoresis,2006,27(13):2782-2786.[2] 兰彦,钱小红.蛋白质组分析中蛋白质分步提取方法的建立[J]. 生物化学与生物物理进展,2001,28(3):415-418.[3] 曾亚. 水稻抗病基因介导的抗病反应中蛋白质表达谱的比较分析[D]. 武汉:华中农业大学,2008.[4] 严顺平. 水稻响应盐胁迫和低温胁迫的蛋白质组研究[D]. 北京:中国科学院研究生院,2005.[5] 赵敏. 水稻两优培九不同氮素处理叶片和籽粒蛋白质组学研究[D]. 福州:福建农林大学,2008.[6] 汪家政,范明.蛋白质技术手册[M]. 北京:科学出版社,2000. [7] WANG W, SCALI M, VIGNANI R, et al. Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds[J]. Electrophoresis,2003,24(14):2369-2375.[8] FAUROBERT M, PELPOIR E, CHA B J. Phenol extraction of proteins for proteomic studies of recalcitrant plant tissues[J]. Methods in Molecular Biology,2006,355:9-14.[9] LI F F,WANG Y Y,LIU G F, et al. Cloning and expression of wheat heat-shockprotein 60(HSP60) gere in E. coli[D]. Agricultural Science & Technology,2010,11(1):5-7.[10] 郭尧君. 蛋白质电泳实验技术[M]. 北京:科学出版社,2005.[11] 丁.萨姆布鲁克, D.W.拉塞尔. 分子克隆实验指南[M]. 第三版.黄培堂,王嘉玺,朱厚础,等译.北京:科学出版社,2002.[12] 王卓. 一个新的烟草糖基转移酶的过表达及相关研究[D]. 武汉:中南民族大学,2008.[13] 王雪,谭艳平,周杰,等. 一个烟草糖基转移酶启动子在转基因烟草植株中的表达分析[J]. 安徽农业科学,2010,38(18):9426-9428.。
植物蛋白质提取方法
一、植物蛋白质提取1. TCA-丙酮法（1）称量一定量的样品置于液氮预冷的研钵中，加少许PVPP，反复加液氮研磨至粉末。
（2）研磨好的样品用10 倍体积（w/V）的10%的TCA-丙酮溶液悬浮，加入0.1 M PMSF、1 M DTT 至终浓度为1 mM PMSF、10mM DTT，超声5 分钟，于-20℃静置6 小时或过夜后，4℃，20000g 离心15 分钟，弃上清。
（3）沉淀用10 倍体积于样品的丙酮溶液重悬浮，于-20℃静置1 小时后，4℃，20000g 离心15 分钟，弃上清。
（4）重复步骤（3）一次。
（5）沉淀用10 倍体积于样品的乙醇/乙醚=1:1 洗，于-20℃静置1 小时后，4℃，20000g 离心15 分钟，弃上清。
（6）重复步骤（3）一次。
（7）取出沉淀真空干燥约5 分钟，除尽有机溶剂。
（8）按10mg 干粉末加200 微升裂解液的比例，加入1 mM PMSF、10mM DTT，超声5分钟，充分溶解1 小时，10℃，35000g 离心30 分钟，上清为所需的蛋白溶液。
（9）用Bradford 法测定蛋白样品的蛋白浓度。
（10）蛋白样品溶液小量分装，-80℃保存。
注意事项：（1）TCA 有利于去除酚类色素等物质，但不利于蛋白的抽提，使用浓度不宜过高，在后面丙酮处理中，尽量除去。
（2）蛋白样品保存：样品浓度不宜过高，建议将高浓度样品适度调整，为避免反复冻融应分装保存，长期保存用-80℃，短期保存用-20℃。
（3）1M DTT：0.1542g DTT 用1ml MilliQ 水溶解，-20℃保存。
（4）0.1M PMSF：0.0174g PMSF 用1ml 异丙醇溶解，-20℃保存。
沉淀蛋白质的常用方法
沉淀蛋白质的常用方法（TCA、乙醇、丙酮沉淀蛋白操作步骤）2010-08-18 15:19TCA-DOCFor precipitation of very low protein concentration1) To one volume of protein solution, add 1/100 vol. of 2% DOC (Na deoxy cholate, detergent).2) Vortex and let sit for 30min at 4oC.3) Add 1/10 of Trichloroacetic acid (TCA) 100% vortex and let sit ON at 4oC (preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottle at 4oC.Be careful, use gloves!!!).4) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see). [OPTION: Wash pellet twice repellet samples 5min at full speed between washes].5) Dry samples under vaccum (speed vac) or dry air. For PAGE-SDS, resuspend samples in a minimal volume of sample buffer. (The presence of some TCA can give a yellow colour as a consequence of the acidification of the sample buffer ; titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.)Normal TCATo eliminate TCA soluble interferences and protein concentration1) To a sample of protein solution add Trichloroacetic acid (TCA) 100% to get 13% final concentration. Mix and keep 5min –20oC and then 15min 4oC; or longer time at 4oC without the –20oC step for lower protein concentration. Suggestion: leave ON if the protein concentration is very low.(preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottle at 4oC.Be careful, use gloves!!!).2) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).3) For PAGE-SDS, resuspend samples in a minimal volume of sample buffer. (The presence of some TCA can give a yellow colour as a consequence of the acidification of the sample buffer ; titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.)Acetone PrecipitationTo eliminate acetone soluble interferences and protein concentration1) Add 1 volume of protein solution to 4 volumes of cold acetone. Mix and keep at least 20min –20oC. (Suggestion: leave ON if the protein concentration is very low).2) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).3) Dry samples under vaccum (speed-vac) or dry air to eliminate any acetone residue (smell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.Ethanol PrecipitationUseful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS1) Add to 1 volume of protein solution 9 volumes of cold Ethanol 100%. Mix and keep at least 10min.at –20oC. (Suggestion: leave ON).2) Spin 15min 4oC in microcentrifuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).3) Wash pellet with 90% cold ethanol (keep at –20oC). Vortex and repellet samples 5min at full speed.4) Dry samples under vaccum (speed vac) or dry air to eliminate any ethanol residue (smell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.TCA-DOC/AcetoneUseful method to concentrate proteins and remove acetone and TCA soluble interferences1. To one volume of protein solution add 2% Na deoxycholate (DOC) to 0.02% final (for 100 μl sample, add 1 μl 2% DOC).2. Mix and keep at room temperature for at least 15 min.3. 100% trichloroacetic acid (TCA) to get 10% final concentration (preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottle at 4oC.Be careful, use gloves!!!).4. Mix and keep at room temperature for at least 1 hour.5. Spin at 4oC for 10 min, remove supernatant and retain the pellet. Dry tube by inversion on tissue paper.6. Add 200 μl of ice cold acetone to TCA pellet.7. Mix and keep on ice for at least 15 min.8. Spin at 4oC for 10 min in microcentrifuge at maximum speed.9. Remove supernatant as before (5), dry air pellet to eliminate anyacetone residue (smell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.10. (The presence of some TCA can give a yellow colour as a consequence of the acidification of the sample buffer ; titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.)Acidified Acetone/MethanolUseful method to remove acetone and methanol soluble interferences like SDS before IEF1) Prepare acidified acetone: 120ml acetone + 10μl H Cl (1mM final concentration).2) Prepare precipitation reagent: Mix equal volumes of acidified acetone and methanol and keep at -20oC.3) To one volume of protein solution add 4 volumes of cold precipitation reagent. Mix and keep ON at -20oC.4) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).5) Dry samples under vaccum (speed-vac) or dry air to eliminate any acetone or methanol residue (smell tubes).TCA-Ethanol PrecipitationUseful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS1) Dilute 10-25μl samples to 100μl with H2OAdd 100μl of 20% trichloroacetic acid (TCA) and mix (prepa ration of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottle at 4oC.Be careful, use gloves!!!).2) Leave in ice for 20min. Spin at 4oC for 15 min in microcentrifuge at maximum speed.3) Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue (pellet may be difficult to see).4) Wash pellet with 100μl ice-cold ethanol, dry and resuspend in sample buffer.5) In case there are traces of GuHCl present, samples should be loaded immediately after boiling for 7 min at 95°C6) (The presence of some TCA can give a yellow colour as a consequence of the acidification of the sample buffer ; titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.)PAGE prepTM Protein Clean-up and Enrichment Kit - PIERCEThe PAGE prep? Kit enables removal of many chemicals that interfere with SDS-PAGE analysis: guanidine, ammonium sulfate, other common salts, acids and bases, detergents, dyes, DNA, RNA, and lipids.PIERCE: #26800 - PAGE prepTM Protein Clean-up and Enrichment Kit (pdf)Chloroform Methanol PrecipitationUseful method for Removal of salt and detergents1) To sample of starting volume 100 ul2) Add 400 ul methanol3) Vortex well4) Add 100 ul chloroform5) Vortex6) Add 300 ul H2O7) Vortex8) Spin 1 minute @ 14,0000 g9) Remove top aqueous layer (protein is between layers)10) Add 400 ul methanol11) Vortex12) Spin 2 minutes @ 14,000 g13) Remove as much MeOH as possible without disturbing pellet14) Speed-Vac to dryness15) Bring up in 2X sample buffer for PAGEReference: Wessel, D. and Flugge, U. I. Anal. Biochem. (1984) 138, 141-143哈哈,我做过这个论文哈!1. 配胶缓冲液系统对电泳的影响？在SDS－PAGE不连续电泳中，制胶缓冲液使用的是Tris－HCL缓冲系统，浓缩胶是pH6.7，分离胶pH8.9；而电泳缓冲液使用的Tris－甘氨酸缓冲系统。
沉淀蛋白质的通用方法(TCA,乙醇,丙酮沉淀蛋白操作技巧步骤)
沉淀蛋白质的常用方法（TCA、乙醇、丙酮沉淀蛋白操作步骤）TCA-DOCFor precipitation of very low protein concentration1) To one volume of protein solution, add 1/100 vol. of 2% DOC (Na deoxycholate, detergent).2) Vortex and let sit for 30min at 4oC.3) Add 1/10 of Trichloroacetic acid (TCA) 100% vortex and let sit ON at 4oC (preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat 4oC.Be careful, use gloves).4) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see). [OPTION: Wash pellet twice with one volume of cold acetone (acetone keep at –20oC). Vortex and repellet samples 5min at full speed between washes].5) Dry samples under vaccum (speed vac) or dry air. For PAGE-SDS, resuspend samples in a minimal volume of sample buffer. (The presence of some TCA can give a yellow colour as a consequence of theacidification of the sample buffer ; titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.)Normal TCATo eliminate TCA soluble interferences and protein concentration1) To a sample of protein solution add Trichloroacetic acid (TCA) 100% to get 13% final concentration. Mix and keep 5min –20oC and then 15min 4oC; or longer time at 4oC without the –20oC step for lower protein concentration. Suggestion: leave ON if the protein concentration is very low.(preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat 4oC.Be careful, use gloves).2) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).3) For PAGE-SDS, resuspend samples in a minimal volume of sample buffer. (The presence of some TCA can give a yellow colour as a consequence of the acidification of the sample buffer ; titrate with 1NNaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.)Acetone PrecipitationTo eliminate acetone soluble interferences and protein concentration 1) Add to 1 volume of protein solution 4 volumes of cold acetone. Mix and keep at least 20min –20oC. (Suggestion: leave ON if the protein concentration is very low).2) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).3) Dry samples under vaccum (speed-vac) or dry air to eliminate any acetone residue (smell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.Ethanol PrecipitationUseful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS1) Add to 1 volume of protein solution 9 volumes of cold Ethanol 100%. Mix and keep at least 10min.at –20oC. (Suggestion: leave ON).2) Spin 15min 4oC in microcentrifuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).3) Wash pellet with 90% cold ethanol (keep at –20oC). Vortex and repellet samples 5min at full speed.4) Dry samples under vaccum (speed vac) or dry air to eliminate any ethanol residue (smell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.TCA-DOC/AcetoneUseful method to concentrate proteins and remove acetone and TCA soluble interferences1. To one volume of protein solution add 2% Na deoxycholate (DOC) to 0.02% final (for 100 μl sample, add 1 μl 2% DOC).2. Mix and keep at room temperature for at least 15 min.3. 100% trichloroacetic acid (TCA) to get 10% final concentration(preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat 4oC.Be careful, use gloves).4. Mix and keep at room temperature for at least 1 hour.5. Spin at 4oC for 10 min, remove supernatant and retain the pellet. Dry tube by inversion on tissue paper.6. Add 200 μl of ice cold acetone to TCA pellet.7. Mix and keep on ice for at least 15 min.8. Spin at 4oC for 10 min in microcentrifuge at maximum speed.9. Remove supernatant as before (5), dry air pellet to eliminate any acetone residue (smell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.10. (The presence of some TCA can give a yellow colour as a consequence of the acidification of the sample buffer ; titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.)Acidified Acetone/MethanolUseful method to remove acetone and methanol soluble interferences like SDS before IEF1) Prepare acidified acetone: 120ml acetone + 10μl HCl (1mM final concentration).2) Prepare precipitation reagent: Mix equal volumes of acidified acetone and methanol and keep at -20oC.3) To one volume of protein solution add 4 volumes of cold precipitation reagent. Mix and keep ON at -20oC.4) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).5) Dry samples under vaccum (speed-vac) or dry air to eliminate any acetone or methanol residue (smell tubes).TCA-Ethanol PrecipitationUseful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS1) Dilute 10-25μl samples to 100μl with H2OAdd 100μl of 20% trichloroacetic acid (TCA) and mix (preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat 4oC.Be careful, use gloves).2) Leave in ice for 20min. Spin at 4oC for 15 min in microcentrifuge at maximum speed.3) Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue (pellet may be difficult to see).4) Wash pellet with 100μl ice-cold ethanol, dry and resuspend in sample buffer.5) In case there are traces of GuHCl present, samples should be loaded immediately after boiling for 7 min at 95°C6) (The presence of some TCA can give a yellow colour as a consequence of the acidification of the sample buffer ; titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.)PAGE prep TM Protein Clean-up and Enrichment Kit - PIERCEThe PAGEprep? Kit enables removal of many chemicals that interfere with SDS-PAGE analysis: guanidine, ammonium sulfate, other common salts, acids and bases, detergents, dyes, DNA, RNA, and lipids.PIERCE: #26800 - PAGE prep TM Protein Clean-up and EnrichmentKit (pdf)Chloroform Methanol PrecipitationUseful method for Removal of salt and detergents1) To sample of starting volume 100 ul2) Add 400 ul methanol3) Vortex well4) Add 100 ul chloroform5) Vortex6) Add 300 ul H2O7) Vortex8) Spin 1 minute @ 14,0000 g9) Remove top aqueous layer (protein is between layers)10) Add 400 ul methanol11) Vortex12) Spin 2 minutes @ 14,000 g13) Remove as much MeOH as possible without disturbing pellet14) Speed-Vac to dryness15) Bring up in 2X sample buffer for PAGEReference: Wessel, D. and Flugge, U. I. Anal. Biochem. (1984) 138, 141-143蛋白质浓缩——方法很全1130徐炉李2011-05-28 14:35楼主蛋白质浓缩——方法很全- 丁香园论坛-医学/药学/生命科学论坛蛋白质浓缩方法总结一个简便的方法你可以试试：找一透析袋，底部扎紧，袋口扎一去底的塑料或玻璃试管，将待浓缩的液体从管口灌入透析袋中，将整个装置挂在冰箱中，或者用电风扇吹，液体干后可再继续加入，直至样品浓缩至所需体积。
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TCA-丙酮蛋白浓缩
TCA protein precipitation protocol
Stock Solutions: 100% (w/v) Trichloroacetic acid (TCA)
recipe: dissolve 500g TCA (as shipped) into 350 ml dH2O, store at RT.
Precipitation Protocol:
1. Add 1 volume of TCA stock to 4 volumes of protein sample.
i.e. in 1.5ml tube with maximum vol., add 250µl TCA to 1.0ml sample.
2. Incubate 10 min at 4°C.
3. Spin tube in microcentrifuge at 14K rpm, 5 min.
4. Remove supernatant, leaving protein pellet intact. Pellet should be formed from whitish,fluffy ppt.
5. Wash pellet with 200µl cold acetone.
6. Spin tune in microfuge at 14K rpm, 5min.
7. Repeat steps 4-6 for a total of 2 acetone washes.
8. Dry pellet by placing tube in 95°C heat block for 5-10 min to drive off acetone.
9. For SDS-PAGE, add 2X or 4X sample buffer (with or without bME) and boil smaple for
10 min in 95°C herat block before loading smaple onto polyacrylamide gel.
TCA蛋白浓缩步骤：
储存液：100%（W/V）三氯乙酸（TCA）
配制：将500g TCA溶解到350ml dH2O中，室温储存。
浓缩步骤：
1.加1倍体积的TCA储存液到4倍体积的蛋白样品中。
如：在1.5ml的离心管中，1ml
样品中加入250ul的TCA。
2.4度孵育10min。
3.14 000rpm 离心5 min。
4.弃上清
5.加入200ul预冷的丙酮洗涤沉淀。
6.14 000rpm 离心5 min。
7.重复步骤4-6两次。
8.95度5-10 min，晾干沉淀以彻底出去丙酮。
9.加入2X或4X样品buffer，煮沸样品10 min，SDS-PAGE
改进版：
TCA蛋白浓缩步骤：
储存液：100%（W/V）三氯乙酸（TCA）
配制：将500g TCA溶解到350ml dH2O中，室温储存。
浓缩步骤：
10.TCA储存液加到样品蛋白中，终浓度为10%，TCA缓慢滴入蛋白样品中；蛋白样品不
易过多，2ml左右即可，使用10ml离心管。
11.冰浴30-60min，时间久一点。
12.14 000rpm 离心5 min。
13.弃上清
14.加入400ul预冷的丙酮洗涤沉淀，（丙酮事先放入-30℃冰箱内冷冻至少半个小时以上）。
15.14 000rpm 离心5 min。
16.重复步骤4-6两次。
17.室温晾干沉淀，不易过干10-15min。
18.加入水溶解，若有结块或不溶可用8M尿素溶解；
19.区样品做跑胶样品处理，跑SDS胶。