沉淀蛋白質(zhì)的常用方法(TCA、乙醇、丙酮沉淀蛋白操作步驟)

沉淀蛋白質(zhì)的常用方法（TCA、乙醇、丙酮沉淀蛋白操作步驟）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 (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 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% 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 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 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 Precipitation Useful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS 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 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/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 (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 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 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 methanol 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 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 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 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, 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 TrisHCl 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, 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蛋白質(zhì)濃縮方法很全1130徐爐李2011-05-28 14:35樓主蛋白質(zhì)濃縮方法很全 - 丁香園論壇-醫(yī)學(xué)/藥學(xué)/生命科學(xué)論壇蛋白質(zhì)濃縮方法總結(jié)一個(gè)簡(jiǎn)便的方法你可以試試：找一透析袋，底部扎緊，袋口扎一去底的塑料或玻璃試管，將待濃縮的液體從管口灌入透析袋中，將整個(gè)裝置掛在冰箱中，或者用電風(fēng)扇吹，液體干后可再繼續(xù)加入，直至樣品濃縮至所需體積。如必要，可事先將待濃縮液用蒸餾水或去離子水透析以去鹽分，然后再按上述方法濃縮，這樣可以避免濃縮后鹽份過(guò)高。此法簡(jiǎn)便易行，我們實(shí)驗(yàn)室常用此法濃縮。濃縮后的液體可以用吸管從試管口吸出，而且透析袋只要不破裂，可以反復(fù)使用?？梢杂胢illipore 公司的amicon ultra15 cenfilter unitetrifugal units。每個(gè)30多元，但是效果非常好。只需要將培養(yǎng)上清加入，然后高速離心后即可直接得到無(wú)菌的濃縮液。對(duì)于新的透析袋有化學(xué)雜質(zhì)，需要處理，為了去除這些雜質(zhì)，通常我們用10mmol/L的碳酸氫鈉，1mmol/L的EDTA溶液煮沸30min，之后用雙蒸水充分沖洗，之后放在EDTA溶液4度保存！防止微生物污染！冷凍干燥或peg20000都可以此種方法就是超濾濃縮，使用方式有的是離心，有的是加壓，有的二者皆用。除了amicon之外，我知道賽多利斯也是很有名的供應(yīng)廠家，就是做天平很出名的那家德國(guó)公司。超濾濃縮這種方式的確太方便了，而且除了有濃縮作用外，還有脫鹽功能，速度又快，比傳統(tǒng)的透析好多了，實(shí)驗(yàn)室用和生產(chǎn)用型號(hào)都能找到。不過(guò)據(jù)介紹，不能反復(fù)用！而且價(jià)格大家也知道了，30元左右，一般一個(gè)包裝25個(gè)，100個(gè)，單個(gè)還不知道賣不賣。根據(jù)中國(guó)國(guó)情，呵呵，我建議沒經(jīng)費(fèi)的實(shí)驗(yàn)室還是不要考慮啦；如果時(shí)間不緊張，不怕花幾天時(shí)間，用25元/米的透析袋一樣可以達(dá)到效果。冷凍干燥的體積當(dāng)然越小越好了，如果你的蛋白很穩(wěn)定那-70度應(yīng)該沒有什么問(wèn)題的，我覺得冷凍干燥時(shí)間長(zhǎng)，如果量不大直接用沉淀的方法濃縮就可以了。濃縮的目的是使體積小這樣快，而體積很大的話冷凍干燥慢，如果你的冷凍干燥機(jī)的真空、泵很好，那么體積大也沒有關(guān)系，但是要保證你的液體的高度別超過(guò)2cm那其實(shí)也是很快的，所以關(guān)鍵看你樣品的體積和你機(jī)器的好壞了?？梢栽囈幌孪旅孢@個(gè)簡(jiǎn)單的辦法：直接向要濃縮的蛋白質(zhì)樣品中加入sephadex G25干粉，待干粉吸水后離心取上清即可，這樣離子強(qiáng)度保持不變，又達(dá)到了濃縮的目的我是用雙蒸水進(jìn)行除鹽的，就是在剛開始的時(shí)候要多換幾次雙蒸水，一般24小時(shí)就可以了。樓上占戰(zhàn)友們所說(shuō)的millipore的超濾離心管我本來(lái)想買的，但考慮到價(jià)格的問(wèn)題就放棄了。將樣品放在透析袋中，注意留出一半的容積，（以免水將透析袋漲破）24小時(shí)后將透析袋放入20%的分子量為10000的聚乙二醇中，一般幾個(gè)小時(shí)就可以了。我買的是進(jìn)口分裝的PEG，感覺不錯(cuò)。希望能對(duì)你有所幫助。三氯乙酸沉淀可能會(huì)使蛋白變性的，那么再用PBS或別的緩沖液都難溶解，所以我覺得如果要想保持蛋白的活性還是別用這樣的方法。電泳樣品當(dāng)然可以，但是要注意你的樣品用緩沖液難溶解，可以直接加電泳上樣緩沖液就可以了，沒有必要再用PBS溶解。三氯乙酸沉淀后，樣品要用丙酮洗，然后要吹干，然后可以重新溶解般可以將樣品放入透析袋，再將周圍撒多點(diǎn)PEG20000，半天能夠濃縮10倍以上如果打算用PEG的話，要選擇好PEG的型號(hào)，某型號(hào)透析袋透過(guò)分子量的大小，還好考慮你的目的蛋白分子量的大小。我用過(guò)PEG2000，回收很方便。它的熔點(diǎn)很低，大約只有50度。就現(xiàn)在的天氣，把它平鋪在磁盤里，自然干燥就行了。不建議大家選用太小分子量的Peg， 要根據(jù)透析帶來(lái)選擇， 我一般是選用透析膜能通過(guò)分子量的兩倍以上的Peg， 如果大家只用來(lái)做Western ， 用蔗糖就行了。1)我們?cè)谧鰏ds-page的時(shí)候，樣品的濃縮是用1M的三氯乙酸，離心以后再加丙酮進(jìn)行清洗，把三氯乙酸除去，如果三氯乙酸沒有除干凈的話，樣品加了LOADING BUFFER以后會(huì)變成黃色，不知道你有沒有出現(xiàn)這種現(xiàn)象？個(gè)人認(rèn)為秀芬蘭旁邊的黑糊糊的一團(tuán)東西可能是樣品中的一些小分子的物質(zhì)。2）有的樣品處理過(guò)程一樣，但寬窄不一，原因可能是樣品中的鹽濃度不一致，鹽濃度過(guò)高會(huì)顯著影響蛋白質(zhì)的電泳，使蛋白質(zhì)條帶明顯變寬，有時(shí)還會(huì)成微笑狀。我的經(jīng)驗(yàn)與老兄不同:1.膠中蛋白條帶寬窄不一原因是:因?yàn)榈鞍捉Y(jié)合電荷量不同!即所加樣品中蛋白含量不同!2.鹽濃度過(guò)高不會(huì)顯著影響蛋白質(zhì)的電泳.我用硫酸銨分級(jí)沉淀時(shí)蛋白未透析.sds-page蛋白條帶沒有變化!你可做個(gè)對(duì)照看一下!取待濃縮樣品 1mL，加入100 uL三氯醋酸，振蕩混勻后于 4oC靜置 30后離心（10000X10 min), 去除上清,沉淀中加入1mL acetone(-20oC預(yù)冷) ，劇烈振蕩混勻后. 10000rpm X5 min,此步可以重復(fù)一次以保證三氯醋酸可以完全除去，最后將管子在室溫放置30分鐘干燥后溶解于緩沖液中電泳分析。如果不是預(yù)冷的丙酮，可以在低溫冰箱中靜置10分鐘即可。看到大家在論壇上經(jīng)常討論這個(gè)問(wèn)題，我也湊湊熱鬧！關(guān)于TCA沉淀蛋白作為SDSPAGE上樣的準(zhǔn)備在本論壇的貼子很多，各種各樣的protocols都有，不過(guò)總結(jié)來(lái)說(shuō)都是含兩個(gè)方面，一是TCA沉淀，二是用丙酮洗去殘留的TCA！也許是大家做上游做的多了，對(duì)于這個(gè)不太熟悉；也許是大家提供的方案太多，仔細(xì)看都不太相同，亂了思緒，我也提提我的看法，希望能理清思緒！TCA沉淀蛋白的方法據(jù)我所知，最早是lowry于1951年發(fā)表在JBC上的一篇題為“PROTEIN MEASUREMENT WITH THE FOLIN PHENOL REAGENT”上用過(guò)的，當(dāng)然這篇文章也就是著名的lowry法定量蛋白的提出篇！隨后在很多文獻(xiàn)，包括現(xiàn)今新的處理蛋白的文獻(xiàn)中也有采用，可見這個(gè)方法如何的被青睞！具體操作方面，不管你用什么4：1的體積比也好，還是摩爾濃度（1M TCA）也好，一般TCA的終濃度在510（W/V）都可以！我從書上節(jié)選了這么樣的一段英文，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 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 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.下面的兩篇文獻(xiàn)可供參考：I. Polacheck and E. Cabib, Anal. Biochem. 117, 311 (1981).2. ENRICO CABIB and ITZHACK POLACHECK,methods in enzymology 104卷，415416丙酮干燥時(shí)間一定不能太長(zhǎng)，否則pellet很難溶的，這是我的經(jīng)驗(yàn)我看最簡(jiǎn)單的是將低濃度蛋白裝入合適透析袋用PEG6000-8000包埋，每10min換一次PEG6000-8000,濃縮至微量體積后再用合適緩沖液透析有可能進(jìn)入的微量小分子PEG，速度很快，我用它作過(guò)病毒的濃縮丙酮沉淀都說(shuō)會(huì)丟失蛋白質(zhì)，可是還有這么多人用沉淀法！1、到底會(huì)丟失多少蛋白質(zhì)？2、為什么會(huì)丟失？3、尿素和其他裂解液的成分溶于丙酮嗎？如果跟著沉淀下來(lái)，會(huì)不會(huì)影響上樣液中各種成分的濃度，進(jìn)而影響聚焦？4、丙酮風(fēng)干不充分，會(huì)影響IEF聚焦嗎？5、一般你們沉淀多久？最少多久？6、丙酮沉淀真的可以去除離子嗎？我的標(biāo)本同樣沉淀，為什么有時(shí)電壓上不去有時(shí)上的去？1,肯定會(huì)丟失蛋白質(zhì),具體丟多少取決于樣品的種類.2,有些蛋白質(zhì)一經(jīng)沉淀之后難以再溶解.3,應(yīng)該不會(huì)跟著沉淀下來(lái),至少絕大部分不會(huì).4,一般5分鐘風(fēng)干足夠了,盡量充分.5,最少2小時(shí)以上以沉淀完全,減少丟失的蛋白質(zhì)。偶常用沉淀過(guò)夜。，可去除大部分離子，但還有一小部分會(huì)跟著沉淀。丙酮中可以加，也可以不加。建議還是加,對(duì)溶解性有好處.-20度沉淀過(guò)夜沒問(wèn)題.我用丙酮沉淀發(fā)酵液的蛋白質(zhì)，發(fā)酵液含有磷酸鹽緩存液，用丙酮沉淀后鹽含量很高。我做了一個(gè)對(duì)照試驗(yàn)，將丙酮直接加在一定濃度磷酸鹽緩存液中，明顯看到白色的沉淀，我感覺丙酮的去鹽不好。謝謝你，我也是因?yàn)橛行岩蛇@個(gè)才發(fā)帖子的。你都用鹽溶液試過(guò)了，事實(shí)勝于雄辯，看來(lái)去鹽最多只是一部分。根據(jù)你的提示，回頭我試試向裂解液中加丙酮，看看尿素等會(huì)不會(huì)沉淀下來(lái)。另外，丙酮不是奪取水分，蛋白才會(huì)沉淀下來(lái)嗎？所以溶于水的成分必然會(huì)沉淀下來(lái)。這是我的理論上的推測(cè)。（供大家討論丙酮沉淀的原理）另外我還覺得，如果裂解液的成分跟著沉淀下來(lái)，會(huì)影響上樣液中各種成分的濃度，進(jìn)而影響聚焦。所以我覺得沉淀應(yīng)該考慮裂解液的成分和濃度。（個(gè)人觀點(diǎn)，供大家討論）建議主任給上面觀點(diǎn)的朋友加分。有鹽的樣品其實(shí)用丙酮淀肯定鹽也會(huì)沉淀下來(lái)的，這取決于鹽的濃度和蛋白的濃度，理論上蛋白的