沉淀蛋白质的常用方法(TCA、乙醇、丙酮沉淀蛋白操作步骤)

1、.沉淀蛋白质的常用方法（TCA、乙醇、丙酮沉淀蛋白操作步骤）TCA-DOC For precipitation of very low protein concentration 1) 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 (

2、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 pel

3、let 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 colou

4、r 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 TCA To eliminate TCA soluble interferences and protein concentration 1) To a sample of protein solution add Trichloroacetic acid (TCA) 100%

5、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,

6、 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 c

7、an 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 Precipitation To eliminate acetone soluble interferences and protein concentration 1) Add to 1 volume of protein solu

8、tion 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 dif

9、ficult 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 Precipitation Useful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS

10、1) 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

11、 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

12、/Acetone Useful method to concentrate proteins and remove acetone and TCA soluble interferences 1. To one volume of protein solution add 2% Na deoxycholate (DOC) to 0.02% final (for 100 l sample, add 1 l 2% DOC). 2. Mix and keep at room temperature for at least 15 min. 3. 100% trichloroacetic acid (

13、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.

14、 6. Add 200 l 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

15、 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/Methanol Useful method to remove acetone and metha

16、nol soluble interferences like SDS before IEF 1) Prepare acidified acetone: 120ml acetone + 10l 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 precipita

17、tion 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

18、 or methanol residue (smell tubes). TCA-Ethanol Precipitation Useful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS 1) Dilute 10-25l samples to 100l with H2O Add 100l of 20% trichloroacetic acid (TCA) and mix (preparation of 100% TCA: 454ml H2O/kg TCA. Maintain

19、 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 100l ice-cold ethanol

20、, 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 95C 6) (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 TrisHC

21、l pH8.5 to obtain the normal blue sample buffer colour.) PAGE prepTM 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, an

22、d 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

23、 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.

24、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。如必要，可事先将待浓缩液用蒸馏水或去离子水透析以去盐分，然后再按上述方法浓缩，这样可以避免浓缩后盐份过高。此法简便易行，我们实验室常用此法浓缩。浓缩后的液体可以用吸管从试管口吸出，而且透析袋

25、只要不破裂，可以反复使用。可以用millipore 公司的amicon ultra15 cenfilter unitetrifugal units。每个30多元，但是效果非常好。只需要将培养上清加入，然后高速离心后即可直接得到无菌的浓缩液。对于新的透析袋有化学杂质，需要处理，为了去除这些杂质，通常我们用10mmol/L的碳酸氢钠，1mmol/L的EDTA溶液煮沸30min，之后用双蒸水充分冲洗，之后放在EDTA溶液4度保存！防止微生物污染！冷冻干燥或peg20000都可以此种方法就是超滤浓缩，使用方式有的是离心，有的是加压，有的二者皆用。除了amicon之外，我知道赛多利斯也是很有名的供应厂家

26、，就是做天平很出名的那家德国公司。超滤浓缩这种方式的确太方便了，而且除了有浓缩作用外，还有脱盐功能，速度又快，比传统的透析好多了，实验室用和生产用型号都能找到。不过据介绍，不能反复用！而且价格大家也知道了，30元左右，一般一个包装25个，100个，单个还不知道卖不卖。根据中国国情，呵呵，我建议没经费的实验室还是不要考虑啦；如果时间不紧张，不怕花几天时间，用25元/米的透析袋一样可以达到效果。冷冻干燥的体积当然越小越好了，如果你的蛋白很稳定那-70度应该没有什么问题的，我觉得冷冻干燥时间长，如果量不大直接用沉淀的方法浓缩就可以了。浓缩的目的是使体积小这样快，而体积很大的话冷冻干燥慢，如果你的冷冻

27、干燥机的真空、泵很好，那么体积大也没有关系，但是要保证你的液体的高度别超过2cm那其实也是很快的，所以关键看你样品的体积和你机器的好坏了。可以试一下下面这个简单的办法：直接向要浓缩的蛋白质样品中加入sephadex G25干粉，待干粉吸水后离心取上清即可，这样离子强度保持不变，又达到了浓缩的目的我是用双蒸水进行除盐的，就是在刚开始的时候要多换几次双蒸水，一般24小时就可以了。楼上占战友们所说的millipore的超滤离心管我本来想买的，但考虑到价格的问题就放弃了。将样品放在透析袋中，注意留出一半的容积，（以免水将透析袋涨破）24小时后将透析袋放入20%的分子量为10000的聚乙二醇中，一般几个

28、小时就可以了。我买的是进口分装的PEG，感觉不错。希望能对你有所帮助。三氯乙酸沉淀可能会使蛋白变性的，那么再用PBS或别的缓冲液都难溶解，所以我觉得如果要想保持蛋白的活性还是别用这样的方法。电泳样品当然可以，但是要注意你的样品用缓冲液难溶解，可以直接加电泳上样缓冲液就可以了，没有必要再用PBS溶解。三氯乙酸沉淀后，样品要用丙酮洗，然后要吹干，然后可以重新溶解般可以将样品放入透析袋，再将周围撒多点PEG20000，半天能够浓缩10倍以上如果打算用PEG的话，要选择好PEG的型号，某型号透析袋透过分子量的大小，还好考虑你的目的蛋白分子量的大小。我用过PEG2000，回收很方便。它的熔点很低，大约只

29、有50度。就现在的天气，把它平铺在磁盘里，自然干燥就行了。不建议大家选用太小分子量的Peg， 要根据透析带来选择， 我一般是选用透析膜能通过分子量的两倍以上的Peg， 如果大家只用来做Western ， 用蔗糖就行了。1)我们在做sds-page的时候，样品的浓缩是用1M的三氯乙酸，离心以后再加丙酮进行清洗，把三氯乙酸除去，如果三氯乙酸没有除干净的话，样品加了LOADING BUFFER以后会变成黄色，不知道你有没有出现这种现象？个人认为秀芬兰旁边的黑糊糊的一团东西可能是样品中的一些小分子的物质。2）有的样品处理过程一样，但宽窄不一，原因可能是样品中的盐浓度不一致，盐浓度过高会显著影响蛋白质的

30、电泳，使蛋白质条带明显变宽，有时还会成微笑状。我的经验与老兄不同:1.胶中蛋白条带宽窄不一原因是:因为蛋白结合电荷量不同!即所加样品中蛋白含量不同!2.盐浓度过高不会显著影响蛋白质的电泳.我用硫酸铵分级沉淀时蛋白未透析.sds-page蛋白条带没有变化!你可做个对照看一下!取待浓缩样品 1mL，加入100 uL三氯醋酸，振荡混匀后于 4oC静置 30后离心（10000X10 min), 去除上清,沉淀中加入1mL acetone(-20oC预冷) ，剧烈振荡混匀后. 10000rpm X5 min,此步可以重复一次以保证三氯醋酸可以完全除去，最后将管子在室温放置30分钟干燥后溶解于缓冲液中电泳

31、分析。如果不是预冷的丙酮，可以在低温冰箱中静置10分钟即可。看到大家在论坛上经常讨论这个问题，我也凑凑热闹！关于TCA沉淀蛋白作为SDSPAGE上样的准备在本论坛的贴子很多，各种各样的protocols都有，不过总结来说都是含两个方面，一是TCA沉淀，二是用丙酮洗去残留的TCA！也许是大家做上游做的多了，对于这个不太熟悉；也许是大家提供的方案太多，仔细看都不太相同，乱了思绪，我也提提我的看法，希望能理清思绪！TCA沉淀蛋白的方法据我所知，最早是lowry于1951年发表在JBC上的一篇题为“PROTEIN MEASUREMENT WITH THE FOLIN PHENOL REAGENT”上用

32、过的，当然这篇文章也就是著名的lowry法定量蛋白的提出篇！随后在很多文献，包括现今新的处理蛋白的文献中也有采用，可见这个方法如何的被青睐！具体操作方面，不管你用什么4：1的体积比也好，还是摩尔浓度（1M TCA）也好，一般TCA的终浓度在510（W/V）都可以！我从书上节选了这么样的一段英文，大家可以看看：We routinely precipitate samples with trichloroacetic acid (TCA) before SDS-PAGE.This concentrates the samples, eliminates a great deal of buffer

33、 variability, and denatures proteinases and substrates in a rapid step that does not allow time for extensive artifactual proteolysis. Typically, a final concentration of TCA of 5% (w/v) at 4C for 10 min will be adequate. The precipitated proteins are recovered by brief centrifugation (10,000g for 1

34、 min, in a benchtop microcentrifuge),and the TCA is poured away. Residual TCA, which would otherwise change the pH of the sample buffer, is removed by repeated gentle washing with acetone or diethyl ether,and after three washes, the protein pellet is redissolved in SDS-PAGE sample buffer.下面的两篇文献可供参考

35、：I. Polacheck and E. Cabib, Anal. Biochem. 117, 311 (1981).2. ENRICO CABIB and ITZHACK POLACHECK,methods in enzymology 104卷，415416丙酮干燥时间一定不能太长，否则pellet很难溶的，这是我的经验我看最简单的是将低浓度蛋白装入合适透析袋用PEG6000-8000包埋，每10min换一次PEG6000-8000,浓缩至微量体积后再用合适缓冲液透析有可能进入的微量小分子PEG，速度很快，我用它作过病毒的浓缩丙酮沉淀都说会丢失蛋白质，可是还有这么多人用沉淀法！1、到底会丢失

36、多少蛋白质？2、为什么会丢失？3、尿素和其他裂解液的成分溶于丙酮吗？如果跟着沉淀下来，会不会影响上样液中各种成分的浓度，进而影响聚焦？4、丙酮风干不充分，会影响IEF聚焦吗？5、一般你们沉淀多久？最少多久？6、丙酮沉淀真的可以去除离子吗？我的标本同样沉淀，为什么有时电压上不去有时上的去？1,肯定会丢失蛋白质,具体丢多少取决于样品的种类.2,有些蛋白质一经沉淀之后难以再溶解.3,应该不会跟着沉淀下来,至少绝大部分不会.4,一般5分钟风干足够了,尽量充分.5,最少2小时以上以沉淀完全,减少丢失的蛋白质。偶常用沉淀过夜。，可去除大部分离子，但还有一小部分会跟着沉淀。丙酮中可以加，也可以不加。建议还是

37、加,对溶解性有好处.-20度沉淀过夜没问题.我用丙酮沉淀发酵液的蛋白质，发酵液含有磷酸盐缓存液，用丙酮沉淀后盐含量很高。我做了一个对照试验，将丙酮直接加在一定浓度磷酸盐缓存液中，明显看到白色的沉淀，我感觉丙酮的去盐不好。谢谢你，我也是因为有些怀疑这个才发帖子的。你都用盐溶液试过了，事实胜于雄辩，看来去盐最多只是一部分。根据你的提示，回头我试试向裂解液中加丙酮，看看尿素等会不会沉淀下来。另外，丙酮不是夺取水分，蛋白才会沉淀下来吗？所以溶于水的成分必然会沉淀下来。这是我的理论上的推测。（供大家讨论丙酮沉淀的原理）另外我还觉得，如果裂解液的成分跟着沉淀下来，会影响上样液中各种成分的浓度，进而影响聚焦。所以我觉得沉淀应该考虑裂解液的成分和浓度。（个人观点，供大家讨论）建议主任给上面观