TCA 蛋白沉淀方法

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三氯乙酸(TCA)沉淀蛋白的原理

三氯乙酸（TCA）重淀蛋黑的本理之阳早格格创做TCA 与蛋黑量之间主要有以下几个圆里的效率:
①正在酸性条件下与蛋黑量产死没有溶性盐. ②动做蛋黑量变性剂使蛋黑量构象爆收改变, 表暴露较多的疏火性基团, 使之汇集重淀. ③随着蛋黑量分子量的删大, 其结构搀纯性与致稀性越大, TCA 大概渗进分子里里而使之较易被实足与消, 正在电泳前样品加热处理时大概使蛋黑量结构爆收酸火解而产死碎片, 而且随时间的延少那一效率愈加明隐. ④电泳图谱隐现,BSA , HSA 单体谱戴有较明隐的展宽局里, 那大概是由于TCA 的分离, 使SDS 与蛋黑量的分离量爆收偏偏好, 进而制成蛋黑量所戴电荷的没有均一性, 制成迁移率的纷歧致.咱们认为正在电泳时使用TCA 对付蛋黑量样品的浓缩或者除盐时, 对付于分子品量大的蛋黑量, 要慎重采用TCA. 对付小分子量蛋黑量的浓缩, 采与TCA 时也有二面需要注意: 一是用TCA 重淀后, 尽管用丙酮实足抽提TCA; 二是样品处理后要尽管举止电泳分解, 免得爆收汇集及断裂, 制成截止分解的禁绝确.。

三氯乙酸沉淀蛋白的原理

三氯乙酸沉淀蛋白的原理TCA沉淀蛋白的原理主要包括以下几个方面：1.酸性沉淀：TCA是一种强酸，可生成强酸性环境。

当TCA加入样品中时，会降低样品的pH值，使其酸性增强。

在强酸性条件下，蛋白质分子上的氨基酸产生负电荷，从而导致蛋白质分子间的静电斥力减弱，蛋白质变得不稳定，易于沉淀。

此外，酸性环境还能够破坏蛋白质分子的氢键、疏水键等非共价键，使其失去原有的空间构象和功能。

2.沉淀蛋白：TCA分子中的三氯甲基基团与蛋白质中的氨基酸产生反应，形成三氯乙酸根离子与蛋白质产生结合，从而使蛋白质发生沉淀。

在酸性条件下，蛋白质的溶解度降低，形成团聚体或出现小颗粒状悬浊物。

通过离心操作，可以将蛋白质沉淀下来。

3.降解非蛋白质杂质：TCA除了沉淀蛋白质，还可以沉淀一些非蛋白质杂质，如核酸和多糖等。

由于核酸和多糖也具有负电荷，其在酸性条件下也容易发生沉淀。

因此，在TCA沉淀蛋白过程中，一些非蛋白质杂质也可以同时被沉淀下来，从而进一步净化蛋白质。

4.蛋白质沉淀的后续步骤：TCA沉淀蛋白后，需要进行一系列的处理步骤来清洗和纯化蛋白质。

常见的处理方法包括冷酒精洗涤、去除杂质的洗涤和再溶解等。

通过这些处理步骤，可以有效清除TCA和其他杂质，最终获得纯净的蛋白质。

总结起来，TCA沉淀蛋白的原理是通过酸性环境和TCA与蛋白质分子中的氨基酸反应，使蛋白质发生沉淀。

在沉淀过程中，TCA还可以同时沉淀一些非蛋白质杂质，从而起到净化蛋白质的作用。

这种方法简便易行，操作方便，广泛应用于蛋白质的提取、纯化和分析等领域。

蛋白沉淀法

蛋白沉淀法蛋白沉淀法是一种常用的分离蛋白质的方法，其原理是利用化学反应使蛋白质沉淀至底部，从而分离出目标蛋白质。

本文将详细介绍蛋白沉淀法的原理、步骤、优缺点以及应用领域。

一、原理蛋白沉淀法的原理基于化学反应，常用的反应剂包括三氯醋酸（TCA）、硫酸铵（AS）、三硝基苯磺酸（TNBS）等。

其中，TCA法是最常用的方法之一。

TCA与蛋白质反应后，会形成一种不溶于水的复合物，从而使蛋白质沉淀至底部。

TCA法的反应方程式如下：TCA + 蛋白质→ TCA-蛋白复合物二、步骤蛋白沉淀法的步骤通常包括以下几个步骤：1. 样品制备：将待分离的样品加入适量的缓冲液中，使其pH值在7左右。

2. 加入反应剂：将反应剂加入样品中，通常加入的量为样品体积的1/10至1/5。

3. 沉淀：将反应液在4℃下静置30分钟至1小时，使蛋白质充分沉淀至底部。

4. 洗涤：用冷乙醇或冷醚洗涤沉淀，去除残余的反应剂和其他杂质。

5. 脱水：将沉淀放入干燥器中，用低温低压的方式将水分脱除。

6. 重溶：用适量的缓冲液将沉淀重溶，得到目标蛋白质。

三、优缺点1. 优点：蛋白沉淀法操作简单，成本低廉，适用于大规模分离蛋白质。

此外，该方法还可以去除大量的杂质和非蛋白质物质。

2. 缺点：蛋白沉淀法的选择性不够高，可能会将多种蛋白质沉淀至底部。

此外，该方法会对蛋白质的结构和功能产生一定的影响，使得蛋白质的活性降低。

四、应用领域蛋白沉淀法广泛应用于生物学、生化学、医学等领域。

其中，最常见的应用包括：1. 分离纯化蛋白质：蛋白沉淀法可以将目标蛋白质从复杂的混合物中分离出来，得到较为纯净的蛋白质样品。

2. 检测蛋白质含量：蛋白沉淀法可以用于检测样品中蛋白质的含量，并进行定量分析。

3. 蛋白质结构研究：蛋白沉淀法可以用于分离蛋白质的亚单位，从而研究蛋白质的结构和功能。

总之，蛋白沉淀法是一种常用的分离蛋白质的方法，其原理简单，操作方便，适用于大规模分离蛋白质。

但是，由于其选择性不够高，会对蛋白质的结构和功能产生一定的影响，因此在具体应用时需谨慎选择。

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。

tca沉淀蛋白的步骤

tca沉淀蛋白的步骤TCA（三氯乙酸）沉淀蛋白是一种常见的蛋白质沉淀方法，可用于提取蛋白质、去除杂质以及富集样品中的蛋白质。

以下是TCA沉淀蛋白的详细步骤：步骤1：样品制备首先需要准备待沉淀的样品，可以是细胞裂解液或组织提取液。

样品可以通过细胞破碎、组织切割等方法进行制备。

确保在操作过程中样品保持在低温状态，以避免蛋白质的降解。

步骤2：沉淀液的制备接下来需要制备TCA沉淀液。

可以通过将三氯乙酸固体溶解在冷的去离子水中来制备沉淀液。

一般来说，使用10%TCA是一个常见的浓度。

将沉淀液存放在低温冷藏条件下。

步骤3：样品混合将样品与相同体积的TCA沉淀液混合。

混合过程中需要确保样品以及沉淀液的温度保持低温状态。

混合均匀后，将混合物放置在低温环境中静置一段时间。

步骤4：沉淀蛋白的收集将沉淀混合物通过离心机进行离心，通常在最高转速离心10-15分钟。

离心后，上清液会被剔除，而蛋白质沉淀会留在离心管底部。

将上清液小心地倒掉，并用10%TCA溶液进行洗涤，以去除残留的杂质。

步骤5：蛋白质的洗涤和溶解将沉淀涂上10%TCA溶液，并轻轻摇动离心管，以确保蛋白质沉淀的洗涤彻底。

然后用冷醋酸酐洗涤蛋白质沉淀，这个步骤可以去除残留的TCA。

步骤6：蛋白质的溶解将洗涤干净的蛋白质沉淀用样品缓冲液（通常是果糖酸和果糖制备的磷酸盐缓冲液）溶解。

可根据需要添加蛋白酶抑制剂或其他添加剂，以保持蛋白质的活性和稳定性。

步骤7：蛋白质的定量和分析使用合适的方法，例如BCA法或Lowry法等，对溶解后的蛋白质进行浓度定量。

浓度定量后，可以进行后续的蛋白质分析，如SDS-凝胶电泳、Western blotting等。

需要注意的是，TCA沉淀蛋白的方法通常适用于大量的样品，对于低蛋白含量的样品，可能需要进行前处理，如浓缩或富集蛋白质。

此外，TCA沉淀过程中需要注意保持低温，以提高沉淀效果和保护蛋白质的完整性。

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。

TCA 蛋白沉淀方法

100％（ｗ／ｖ）三氯乙酸得配制方法：500g三氯乙酸用227ml水来溶解,所得溶液即１00％三氯乙酸溶液.避光,4度保存.(pｒeｐａｒaｔiｏn of 100%TCA: 454ml H2O/kg TCA、Ｍaｉntain ｉn ｄarkｂoｔtｌeaｔ4ｏＣ、Ｂeｃａrｅfｕｌ，use gloｖes！！！)、培养基上清直接电泳跑出来得条带经常很难瞧，可以ＴCA沉淀浓缩后跑电泳，一般表达量大于1ｍg/ml可以瞧到明显条带，这就是我用得TCA沉淀方法，效果很好：1、菌液1０0０0g，离心５分钟，收集表达上清。

2、取500－１000ul上清于EP管中，加入1/9体积得10０％TCＡ，颠倒10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，除去残余在管口得液体。

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，如果管壁还残留有液体，可以吸水纸吸掉。

可以改成室温或用电吹风，关键就是除去管底与管壁残余液体.6、15000ｇ，离心10－20分钟,用20ｕl枪头尽量吸去管底残余得液体,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，确认管壁与管底没有液体残留。

8、加入2０－５0ul Lｏadｉｎg ｂｕｆｆer，9５度加热１0nｉm，一般沉淀会自动溶解,如果不溶,用手指轻弹管壁或用２0ul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。

如果蓝色得Loadinｇbuｆｆer 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,而且不影响电泳效果。

或者第５步与第6步改为丙酮洗：5、加入200ul冰冷得丙酮，用手指轻弹EP管,洗去管底与管壁残余得TCA.6、１50００g,离心10－20分钟，倒掉上清,将EP管倒扣在吸水纸上轻轻控几下,除去残余在管口得液体.ＴＣA—DＯCFｏｒ pｒｅｃiｐiｔation of ｖeｒy ｌoｗ proteiｎ cｏncenｔraｔioｎ1) Ｔo oｎe volｕme of pｒotｅinｓolution,add 1／10０vｏl、of ２％DOC (Na deoxychｏｌａte, ｄeｔerｇeｎt）、２）Vorｔex anｄlet sｉt fｏr 30min ａt 4ｏC、ﻫ3)Adｄ１/10 oｆTrichloｒｏaceｔi ｃacid(ＴCA）100％vortex anｄｌet siｔON at 4oC (prepaｒation oｆ100% TC Ａ：454ｍｌＨ2O/kｇTCA、Mａinｔａin ｉｎdark bottlｅaｔ4oC、Be carｅful，ｕse ｇlｏves!！!）、４）Sｐin 1５ｍiｎ4oC ｉn microｆｕｇｅat maxｉmum speed（1５０00ｇ）、Ｃarefu ｌlｙdｉschａrge ｓupernaｔant aｎd retain the pellet: drｙtｕbe by ｉｎverｓｉon ｏｎtissuｅpapｅｒ（pellet may bｅｄifficult to see）、[ＯPTION: Wasｈｐelｌeｔtwi ｃe with ｏne vｏlumｅof cold ａｃｅtone（aｃｅｔone keep aｔ–20oC）、Ｖor ｔex and repeｌｌｅt saｍplｅs 5ｍin aｔｆull sｐeed ｂeｔｗeｅｎwａｓhes］、５）Dry ｓaｍｐles uｎder vaccuｍ（ｓｐｅｅd vａｃ) orｄryａｉr、Fｏr ＰAGＥ—SDS, resuspｅｎd sａmｐlｅs in ａminimal voｌuｍｅof saｍpleｂｕffｅｒ、(Tｈe pｒesence of some TＣＡcan giｖｅa yeｌｌow ｃｏｌour ａs aｃonｓeｑｕe ｎcｅof tｈe aciｄificａtioｎof the sample buｆｆer; titｒatｅwｉth 1N NaOH ｏr 1M TrｉｓＨCｌpH8、５ｔｏｏbｔain the normal blue sａmplｅｂuｆfｅr colｏur、)Nｏrmａl TCＡﻫTo eliminateＴCA soluble iｎtｅｒferｅnces and ｐrｏteｉn coｎceｎtr ａtion1) Ｔo a sampｌe oｆpｒotein soluｔｉｏｎaｄd Ｔrichlorｏacetic aｃｉd（TCA) 100% to get13％finａl cｏncentｒation、Ｍix and keep 5min–20oCａnd tｈｅn 15min ４oC；or lｏngeｒtime at4ｏC wｉthｏuｔthe –2０oC sｔｅｐfoｒlower ｐrotｅin cｏnceｎｔration、Ｓuggestiｏn: lｅave ON iｆｔhe prｏteiｎcoｎcｅntrａtｉon iｓvｅrｙlow、ﻫ（pｒeｐaration of1０0％ＴＣA：454ml H2O／kｇＴＣA、Mainｔａin in dａｒｋbotｔlｅat 4ｏC、Ｂe carefｕl，use glｏves!!!)、2) Spin 1５min 4oC iｎmicｒofuge at mａｘiｍｕｍspeed (150０0g)、Carｅfully dｉｓcharge sｕpｅrnaｔaｎt and retａin ｔhe pellet: dry tubｅｂy inｖｅｒsion on tiｓsue papｅr （pellｅt may be difficｕlt tｏsｅe）、ﻫ3)Fｏr PAＧＥ—ＳＤS,ｒesuspenｄsampleｓiｎa minimal volumeｏf sａmple buｆfer、（Thｅpreｓence of some TCA can give aｙeｌlow cｏloｕｒasａcoｎｓｅquencｅｏf thｅacｉdificatiｏn oｆｔhe sampｌe bｕfｆｅr ; tiｔrａｔe with 1N NaOH or １M TrisＨCｌpH8、5 to obtａｉｎthe nｏrmal ｂlue samｐｌｅbuffｅｒcｏlouｒ、) ﻫAcetone PｒecipitationﻫTo eliminate acetone soｌｕble ｉntｅｒfｅrenceｓand ｐroteiｎcoｎcentratｉoｎ1) Ａｄd ｔo1vｏｌｕme ｏf ｐroteｉn ｓolｕtion 4 ｖoｌumeｓof ｃolｄａcetｏne、Mix aｎｄkeeｐaｔleast 20min –20oＣ、（Suｇgｅstion：leave OＮiｆthe pro ｔein conceｎtratioｎｉs very lｏｗ）、ﻫ2) Ｓpｉｎ15ｍiｎ４oC ｉｎmicrｏfugｅat maximuｍsｐeed （15000g)、Carｅｆulｌy ｄischarge suｐernaｔant and retaｉn tｈe pellet：ｄｒy ｔube byｉｎｖersｉoｎon ｔissuｅpａper （pellｅｔmayｂe dｉfficult to ｓee）、ﻫ3) Ｄｒy ｓａmpｌｅｓｕnｄer vａcｃｕｍ(speeｄ-vaｃ）ｏr ｄｒy ａir tｏeliｍｉnatｅａnｙaｃetｏｎe ｒesidue (smｅll ｔuｂeｓ)、Fｏr PAGＥ—SＤS, resuｓpend saｍples ｉn a ｍｉnimal vｏluｍe of samｐｌe ｂuｆfer、EthanoｌPrecｉｐitaｔiｏnＵseｆｕl methoｄto conｃenｔraｔｅproｔｅiｎｓanｄremoval of Ｇｕａｎiｄine Hydrocｈloｒide bｅfｏre ＰＡGE-SDS１)Aｄd ｔo 1 vｏlume oｆpｒoteiｎsolution 9 ｖoｌｕmes ｏf ｃold Ethanol1０0％、Mix andｋeｅp at leaｓt 10miｎ、at –２0ｏＣ、（Suggesｔiｏn: leave ＯN)、2）Sｐin 1５ｍin 4ｏC in micrｏcｅntrifｕge aｔmaximuｍｓpeeｄ(1５０0０g)、Carefully dｉscharｇe supｅｒｎataｎｔanｄｒetain the pellet: dｒy ｔuｂe by inｖersion oｎtissue paｐｅr(pelｌet ｍａｙbe diｆficult to sｅe）、３）Wash pelｌet with 90％cｏld eｔｈanol（keeｐａt–2０oC)、Ｖortex ａndrepelｌet samｐｌes ５mｉｎａt fuｌl speed、ﻫ４）Dry sａｍplｅs under vaccum (ｓpeedｖac) or drｙａir toｅliｍinａte ａny ethanoｌｒｅｓiduｅ(smell ｔｕｂes）、Ｆor PAGE－SDS, reｓuspenｄsａmples iｎ a miｎimal volｕme of sample buffer、ﻫTCＡ-DＯＣ／AcetoｎeﻫUseful mｅthoｄto concｅｎtrate ｐrｏteiｎs and ｒｅmoｖｅacetｏne aｎd TCA ｓolｕble inｔｅrfereｎces1、To oｎe vｏlume ｏｆｐｒotｅｉｎsoluｔion adｄ2％Ｎａdeoｘycｈolate （DO Ｃ) to 0、02% final（for１0０μl sａmple, aｄd 1 μｌ2% DＯＣ)、2、Mix ａnd keｅｐat ｒoｏm tｅmperａture for ａｔleast 15 min、3、100% triｃhｌｏroacetic ａcｉｄ(ＴCA) tｏget 10% fiｎａl concｅｎtratｉoｎ（pre ｐaration ｏｆ10０%TＣA:45４ml Ｈ2Ｏ/kｇTＣＡ、Mａiｎtaｉｎiｎdaｒk bottl ｅａt 4oC、Be carｅful, usｅgloves！!!）、ﻫ４、Mｉx anｄkeｅp at room teｍpｅｒature foｒat least 1hour、ﻫ5、Spiｎａt 4ｏC fｏｒ１0 min, ｒｅmoｖｅsｕpernａtａn ｔａnｄrｅtａin tｈe pｅllet、Ｄry tube by inverｓｉon on tissuｅpａpｅr、ﻫ6、Aｄd200 μl of ice cold ａcetｏne toＴCA pellet、ﻫ7、Mix aｎd keeｐoｎiｃe ｆorａt leas ｔ１5 min、8、Spin at ４oC ｆor10 mｉn in miｃrocenｔｒifuge at ｍａｘｉmｕm spｅｅd、9、Reｍove ｓｕpｅrnａｔａｎt as befoｒe(5）, dry airｐellｅt tｏelimiｎａte anyａcｅｔoｎe ｒesidue （smell tuｂeｓ）、For PＡGE—SDS，rｅsusｐenｄsaｍples ｉｎａｍinimａl volｕme of sａｍpleｂuffeｒ、ﻫ10、(Ｔｈｅpresenｃe oｆsｏｍｅＴCA can givｅａyellow coloｕr aｓ a consequence oｆthe acｉdificaｔiｏｎof ｔhe saｍple bｕffeｒ; ｔｉtｒaｔe wｉｔh 1N ＮaOH or 1M ＴrisＨCl pH８、５to ｏbt ａin tｈe ｎorｍａl blue ｓampｌe bｕffeｒcｏlouｒ、)Aciｄiｆiｅd Aceｔone／ＭethａｎｏｌﻫUｓefｕlｍetｈod to rｅｍove acetoｎe and methanolｓoluｂle inｔｅrｆerencｅs like SDS befoｒe IＥF1）Prepare aｃidiｆied acetｏｎe: １２0mｌacetone + １0μl HCl （1ｍＭfｉnaｌco ｎcｅnｔration）、2) Pｒepare precipitation reagent: Mix ｅquａｌvolumeｓof acidifｉed ａcetoｎe aｎdmｅthanoｌand keｅp ａｔ—20ｏC、３）Tｏone volume of proteｉn solution add 4vｏlｕmes of ｃolｄpreciｐitation reag ｅｎt、Miｘand kｅep ＯＮat—2０oC、4）Ｓｐin １5min 4ｏC in microfuｇe aｔｍaxiｍuｍsｐｅｅd （１5000g）、Cａrefull ｙdischaｒge ｓupeｒｎatant and reｔain thｅｐeｌlet：dry tuｂe by inversioｎon tis ｓｕe paper (ｐellｅt mａｙｂe ｄifｆicultｔo seｅ）、ﻫ５)Ｄry sampｌｅｓｕnder vaｃcuｍ(ｓｐeed-vac）orｄry air tｏelｉmiｎａte aｎy ａcｅtｏne ｏr mｅthａnｏl resｉｄue （smell tｕbes）、TCA—Ｅtｈanol PｒｅcipitationUｓeｆul methｏｄto coｎcentraｔe prｏｔeinｓand rｅmovaｌof Guａｎidiｎe Hydｒｏchｌoriｄe bｅfoｒｅＰAGＥ—ＳDＳ１）Ｄilｕte１０—25μl sａｍpleｓtｏ100μl with Ｈ2OAdd１0０μl oｆ２0％tｒicｈloｒoacｅtｉｃaｃｉd(TCA) ａnd mｉx（preparation of 100％TCA：45４mｌH2Ｏ/ｋg TCA、Maintaｉn iｎdark bｏｔtｌeat４ｏC、Ｂe caｒeful, use gｌoves!！!）、ﻫ2）Ｌeavｅｉn ice for ２０ｍiｎ、Spｉn at ４oC for 15 min in ｍｉcrocentrifuge aｔｍaxｉｍuｍspｅeｄ、ﻫ3）Caｒｅfully ｄischａrge s ｕpｅｒnatanｔand retaiｎthe pellet：dry tuｂｅby inveｒsion on tissue（pellet ｍ4）Wash ｐellｅｔwith 100μlｉce—cold etｈanol，ａy be difｆｉcｕlt toｓee)、ﻫｄry and reｓusｐend in sａmｐｌe buffer、5）Ｉn caｓeｔhere are traces of GuHＣl prｅｓent,samｐles should bｅloadedｉmmeｄｉａteｌy afｔer boilｉng ｆor ７min at 95°C ﻫ6）(Tｈe pｒeｓｅnce of some TCA ｃａn ｇiｖe a yellow ｃolour as a ｃｏnｓequｅnce ｏf the acｉdifｉｃａｔion of ｔhｅsamｐle buffer ；ｔｉtrate ｗｉtｈ１N ＮaOH oｒ1M TriｓHCl ｐH8、5 to obtain ｔｈe normal bluｅsample ｂｕffｅr ｃolour、）ﻫPAGE preｐTM Pｒｏteｉn Cleａn-up and EnricｈmｅnｔＫit — PＩＥRCEＴｈe PAGEｐreｐ？Kiｔenａbles rｅmoval oｆｍany chemicals tｈa ｔiｎterｆｅre ｗｉth ＳDＳ－PAＧEａｎａlyｓｉｓ：gｕａniｄｉne，ammｏniｕm sulfaｔe, other mon sａlｔs, acidｓanｄbases，detergenｔs，dyeｓ, ＤNＡ,RNA，aｎd liｐids、ＰIＥＲＣE：#26800- ＰＡGＥprｅｐTMＰroteiｎCｌean—up and EｎｒicｈmｅntＫit（ｐｄｆ）ＣhloｒｏｆorｍMethanol PrｅcｉｐitationﻫUseful mｅthｏｄfoｒReｍovaｌof s ａlｔand deterｇenｔs1)To saｍple of ｓtartiｎｇvolumｅ100ul2) Add 400 ul methanol3）Ｖoｒtex wellﻫ4）Ａdd 100 ｕl cｈlorｏform5) Vortexﻫ6）Add30０uｌH2O7）Vortexﻫ8）Spin 1 miｎuｔe 14,0０00ｇ9）Remｏve top aqueouｓlayｅr (protｅin is betweeｎlaｙers）10) Ａdd 4０0ul meｔhanoｌﻫ１1）Voｒtex１2）Spin 2 minｕtes14,000 g１3）Ｒeｍovｅas mucｈMeOH ａｓpｏsｓｉbｌe ｗitｈｏut dｉｓturb14)Speeｄ—Vaｃtｏｄrynessｉng pelleｔﻫ15）Ｂring uｐｉｎ2X samｐle buｆfeｒfｏrＰAGE。

蛋白沉淀方法汇总

TCA沉淀法
1、取500ul蛋白溶液，加入等体积预冷的20%TCA溶液，立刻混匀。

2、冰上放置30分钟。

3、4℃，13200r/min离心15分钟。

4、小心去除上清液，冷冻干燥沉淀，约1小时。

5、加入5ul PBS溶解沉淀，280nm测定蛋白质浓度。

三氯乙酸（TCA）沉淀蛋白质主要基于以下几个方面的作用：在酸性条件下TCA 能与蛋白质形成不溶性盐；TCA作为蛋白质变性剂使蛋白质构象发生改变，暴露出较多的疏水性基团，使之聚集沉淀。

丙酮沉淀法
1、在-20℃预冷丙酮溶液。

2、500ul蛋白液放置于5ml管中，加入3ml预冷丙酮，迅速混匀，-20℃放置2小时以上或过夜。

3、4℃，13200r/min离心15分钟。

4、小心弃去上清液，注意勿接触沉淀。

加入100ml冷的90%丙酮洗涤沉淀，4℃，13200r/min离心5分钟。

5、重复上一步再次洗涤沉淀。

6、弃去上清液，空气中干燥沉淀15-30分钟。

7、加入适量PBS溶解蛋白质沉淀。

丙酮沉淀蛋白质时，必须在0-4℃低温下进行。

蛋白质被丙酮沉淀后，应立即分离，否则蛋白质会变性。

硫酸铵沉淀法适合从大量粗制品中浓缩和纯化蛋白，不适合咱们用。

TCA沉淀法

TCA沉淀法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1mg/ml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10000g,离心5分钟，收集表达上清。

2.取500-1000ul上清于EP管中，加入1/9体积的100％TCA，颠倒10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，如果管壁还残留有液体，可以吸水纸吸掉。

可以改成室温或用电吹风，关键是除去管底和管壁残余液体。

6.15000g,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。

8.加入20－50ul Loading buffer，95度加热10nim，一般沉淀会自动溶解，如果不溶，用手指轻弹管壁或用20ul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，而且不影响电泳效果。

或者第5步和第6步改为丙酮洗:5.加入200ul冰冷的丙酮，用手指轻弹EP管，洗去管底和管壁残余的TCA。

6.15000g,离心10－20分钟,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。

TCA沉淀法定量DNA1. Dilute radioactive sample to a 100 ml volume2. Spot 5 ml of the sample onto the center of a 2.4 cm Whatman GF/C glass-fiber disc.3. Mix 5 ml of the sample with 100 ml Salmon sperm DNA (50 mg in 20 mM ED TA).4. Add 5 mL ice cold 10% Trichloroacetic acid (TCA). Mix well and incubate on ice for 15 min.5. Filter the solution through a separate GF/C glass-fiber disc.6. Wash the filter 6 times with 5 mL ice-cold 10% TCA.7. Wash filter once with 5 mL 95% Ethanol.8. Dry both filter under a heat lamp.9. When dry, count each in a scintillation counter.10. The first filter measures total radioactivity. The second filter measures radioactiv ity incorporated into DNA fragments greater than 20 nucleotides in length.。

tca中底物水平磷酸化的步骤

tca中底物水平磷酸化的步骤
TCA（三氯乙酸）沉淀法是一种用于蛋白质沉淀和富集的常用方法。

在进行TCA沉淀的过程中，蛋白质会被沉淀下来，而底物的水平磷酸化则可以通过以下步骤实现：
1. 细胞裂解，首先需要将含有底物的细胞或组织进行裂解，以释放细胞内的蛋白质和其他生物分子。

2. 底物富集，接下来，可以使用特定的抗体或亲和层析柱等手段来富集含有磷酸化底物的蛋白质。

这一步骤可以帮助我们将感兴趣的磷酸化蛋白质从混合物中分离出来。

3. TCA沉淀，将富集后的蛋白质溶液加入三氯乙酸（TCA），通过TCA的沉淀作用，蛋白质会沉淀下来，而底物的磷酸化状态也会得到保留。

4. 洗涤，沉淀后的蛋白质需要进行洗涤，以去除多余的TCA和其他杂质。

5. 蛋白质溶解，最后，沉淀的蛋白质可以通过加入适当的蛋白
质溶解液来溶解，以便进行后续的分析，如免疫印迹、质谱分析等。

需要注意的是，TCA沉淀法虽然可以用于蛋白质的富集和分离，但在进行实验时需要注意操作细节，以确保底物的磷酸化状态不受
到影响。

另外，实验中还需要使用一些辅助试剂和设备，如磷酸化
特异性抗体、低温离心机等，以确保实验的准确性和可靠性。

沉淀蛋白质的常用方法

沉淀蛋白质的常用方法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 the acidification of the sample buffer ; titrate with 1N NaOH or 1M TrisHClpH8.5 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 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 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 before1) 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 interferences 1. 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 aceton e + 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 before1) Dilute 10-25μl samples to 100μl with H2OAdd 100μl of 20% trichloroacetic acid (TCA) and mix (pre paration of 100% TCA: 454mlH2O/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 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 KitChloroform 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) Vortex 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。

TCA 蛋白沉淀方法

100％（w/v）三氯乙酸的配制方法：500g三氯乙酸用227ml水来溶解，所得溶液即100％三氯乙酸溶液。

避光，4度保存。

(preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat 4oC.Be 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1mg/ml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10000g,离心5分钟，收集表达上清。

2.取500-1000ul上清于EP管中，加入1/9体积的100％TCA，颠倒10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，如果管壁还残留有液体，可以吸水纸吸掉。

可以改成室温或用电吹风，关键是除去管底和管壁残余液体。

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。

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，而且不影响电泳效果。

或者第5步和第6步改为丙酮洗:5.加入200ul冰冷的丙酮，用手指轻弹EP管，洗去管底和管壁残余的TCA。

举例说明蛋白质沉淀的方法

举例说明蛋白质沉淀的方法蛋白质沉淀是分离和浓缩蛋白质的常用方法之一。

通过沉淀，我们可以去除其他干扰性物质，提高我们对目标蛋白质的纯度和浓度。

在本文中，我将为您介绍几种常用的蛋白质沉淀方法，并说明它们的原理和适用范围。

1. 酸性沉淀法：酸性沉淀法是通过在酸性条件下，由于蛋白质的等电点和溶剂中pH 的变化，蛋白质从溶液中聚集并沉淀出来的方法。

这种方法适用于大部分蛋白质，特别是以阴离子方式存在于生物体内的蛋白质。

常用的酸洗涤沉淀剂有三氯醋酸（TCA）和三硝基酸（TNP）等。

2. 盐沉淀法：盐沉淀法是利用高浓度盐溶液与蛋白质发生作用，使蛋白质产生相互作用并沉淀出来的方法。

在高浓度盐溶液中，离子会与蛋白质形成盐桥，并使其失去溶解性。

常用的盐沉淀剂有硫酸铵和饱和硫酸铵等。

3. 有机溶剂沉淀法：有机溶剂沉淀法是通过有机溶剂与蛋白质作用，改变蛋白质的水合作用，使其失去溶解性并沉淀出来的方法。

常用的有机溶剂有丙酮、醇和醚等。

有机溶剂沉淀主要适用于一些具有疏水性的蛋白质。

4. 高温沉淀法：高温沉淀法是通过加热溶液来使蛋白质失去溶解性并沉淀出来的方法。

加热可以改变蛋白质的结构，使其发生凝聚而沉淀出来。

这种方法适用于一些对热稳定的蛋白质。

还有一些其他的蛋白质沉淀方法，如有机相分配法、冷冻沉淀法、醇沉淀法等，它们适用于特定的蛋白质类型或实验条件。

总结回顾：通过以上介绍，我们可以看出蛋白质沉淀是一种重要的蛋白质分离和纯化方法。

在实际应用中，我们可以根据不同的蛋白质性质和实验需求选择合适的蛋白质沉淀方法。

酸性沉淀法和盐沉淀法适用于大部分蛋白质的分离和纯化，有机溶剂沉淀法适用于一些具有疏水性的蛋白质，高温沉淀法适用于热稳定的蛋白质。

还有其他几种沉淀方法可根据实验需要选择使用。

个人观点和理解：作为一位文字写手，我认为蛋白质沉淀是生命科学研究中非常重要的技术手段。

通过蛋白质沉淀，我们可以有效地提取和分离蛋白质，从而更深入地研究其结构和功能。

沉淀蛋白质的常用方法(TCA、乙醇、丙酮沉淀蛋白操作步骤)

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 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 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 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 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 –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 % 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 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 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-SDSO1) Dilute 10-25μl samples to 100μl with H2Add 100μl of 20% trichloroacetic ac id (TCA) and mix (preparation of 100% TCA: 454ml HO/kg TCA. Maintain in dark bottleat careful, use2gloves).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 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 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。

三氯乙酸沉淀蛋白原理

三氯乙酸沉淀蛋白原理三氯乙酸（TCA）沉淀法是一种常用的蛋白质沉淀方法，常用于分离和富集蛋白质。

其原理主要是通过使用三氯乙酸将蛋白质转化为不溶性，从而使其沉淀下来。

下面将详细介绍该方法的原理及其在蛋白质研究中的应用。

首先，三氯乙酸是一种强酸，可以将蛋白质的氨基酸上的羧基质子化，使其带正电荷。

这些带正电荷的氨基酸会相互吸引，从而形成蛋白质聚集体。

同时，三氯乙酸还会降低水的溶解能力，使蛋白质在溶液中变得不稳定。

这样一来，蛋白质会发生凝固并沉淀出来。

其次，蛋白质的溶解度可以受到多种因素的影响，包括溶液的pH值、离子浓度、蛋白质浓度以及人工添加的盐类等。

在TCA沉淀法中，正是通过调节上述因素来促进蛋白质的沉淀。

一般来说，当pH值降低到低于蛋白质的等电点时，蛋白质会开始凝固。

由于三氯乙酸可使溶液的pH值降低，因此这一方法通常在较低的pH条件下进行。

在实际应用中，TCA沉淀法可以用于分离和富集目标蛋白质，去除杂质和其他干扰物。

一般而言，TCA沉淀法比较适用于富含碱性氨基酸的蛋白质，因为这些蛋白质具有较低的等电点，易于在低pH条件下沉淀。

此外，TCA沉淀法还可以去除一些亲水性物质，例如核酸、多肽等，因为这些物质在低pH条件下往往不能与蛋白质结合，从而被沉淀掉。

在某些情况下，TCA沉淀法还可以用于蛋白质的定量。

由于沉淀的蛋白质可以被溶解，并通过一定的测试方法来定量，因此可以根据溶解后的蛋白质浓度推测沉淀前的蛋白质浓度。

不过，需要注意的是，TCA沉淀法的定量结果可能受到一些因素的影响，例如沉淀后的蛋白质可能受到一定程度的降解。

总结起来，TCA沉淀法是一种常用的蛋白质沉淀方法。

其原理主要是通过使用三氯乙酸将蛋白质转化为不溶性的形式，从而使其沉淀下来。

在实际应用中，TCA 沉淀法可以用于分离和富集目标蛋白质，并去除杂质和其他干扰物。

此外，TCA 沉淀法还可以用于蛋白质的定量。

然而，需要注意的是，在使用TCA沉淀法时需要根据样品特性和实验目的进行条件优化，以获得最佳的沉淀效果。

TCA-丙酮沉淀法浓缩蛋白

TCA-丙酮沉淀法浓缩蛋白TCA-丙酮蛋白浓缩TCA protein precipitation protocolStock 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 he at 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 for10 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。

植物蛋白提取

植物全蛋白提取方法：TCA丙酮沉淀法、Tris-HCl法、Trizol沉淀法提取法。

1TCA丙酮沉淀法基于蛋白在酸或疏水条件下变性使蛋白浓缩并去除污染物原理的TCA丙酮沉淀法，最早用于小麦蛋白的提取，是目前提取植物蛋白的常用方法之一。

具有降低次生代物质的干扰、减少蛋白降解等优点。

TCA能有效地抑制蛋白酶对蛋白质的水解作用，保证在制样过程中蛋白质不被降解；丙酮溶液能除去样品中的酚类及色素等干扰物质，同时实验过程中采用的高速离心办法能较好地去除多糖的影响。

然而该方法的一个最大缺点是蛋白质很难重新溶解，而且样品中的非蛋白成分很难除去，可能会丢失膜蛋白和疏水性蛋白，导致2-DE图谱上有明显的横纵条纹。

在研磨样品时加入聚乙烯吡咯烷酮(PVP )或交联聚乙烯基吡咯烷酮(PVPP )用来吸附样品中富含的酚、醌类物质。

它们能通过疏水键与酚类形成复合物，离心可以去除该复合物。

然而，TCA丙酮沉淀法中与蛋白共沉淀的污染物在随后的有机溶剂清洗步骤常难以去除，可以通过振荡和延长蛋白沉淀在裂解缓冲液中温育时间的方法来增加蛋白的溶解能力。

在提取的过程中同时加入了TCA、B-巯基乙醇及DTT 3种药剂可以更好的抑制蛋白质的水解及去除干扰物质。

TCA丙酮提取法耗时少且容易操作，一般作为植物蛋白提取的初始方案，该方法常用于幼嫩组织中蛋白的提取，对更为复杂的植物组织该方法并非最佳选择。

但该方法还是在植物蛋白的提取中占有重要位置，很多木本植物的样品应用该方法效果很好，如鹅掌楸叶片、巴东木莲的雌蕊柱头、槟榔叶片、银杏叶片及枝条、茶树叶片及芽、红豆杉的愈伤组织、石斛叶片等。

草本植物中的大豆叶片、生菜叶片、黄瓜叶片、番茄子叶、龙胆花芽、灰木相思叶片等应用该方法都获得了较清晰的2-DE图谱。

TCA protein precipitation protocolStock 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.1. e. in 1.5ml tube with maximum vol., add 250讥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 200^l 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 for10 min in 95°C herat block before loading smaple onto polyacrylamide gel.2Trizol沉淀法与TCA丙酮沉淀法相比，Trizol沉淀蛋白质的方法可有效地除去色素、酚类等干扰电泳的化学物质，特别是对植物样品中高丰度蛋白Rubisco1,5-二磷酸核酮糖羧化酶/ 加氧酶(Ribulose-1,5-bisphosphate carboxylase/oxygenase，通常简写为RuBisCO)。

tca沉淀蛋白的步骤

tca沉淀蛋白的步骤TCA沉淀蛋白的步骤引言：TCA（三氯乙酸）沉淀法是一种常用的蛋白质沉淀方法，通过沉淀蛋白质可以达到分离、富集和纯化蛋白质的目的。

本文将介绍TCA 沉淀蛋白的步骤及相关注意事项。

一、样品制备1.1 选择合适的样品：样品可以是细胞提取物、组织提取物或其他含有蛋白质的溶液。

1.2 样品浓度调整：为了获得较好的沉淀效果，样品的浓度应适中，通常在0.5-5 mg/mL之间。

1.3 样品处理：如果样品中含有酶活性，可以在样品制备过程中加入抑制剂（如PMSF）来保护蛋白质的完整性。

二、TCA沉淀2.1 加入TCA：将1 mL的样品加入10% TCA溶液中，比例通常是1:4（样品：TCA溶液）。

2.2 混匀：轻轻地旋转样品管或反复倒置，使样品和TCA溶液充分混合。

2.3 孵育：将混合液在4℃下孵育30分钟，促使蛋白质与TCA结合形成沉淀。

2.4 离心：使用高速离心机将样品离心10分钟，以沉淀蛋白质。

三、洗涤沉淀3.1 去除上清液：将上清液完全倒掉，避免上清液中的杂质污染沉淀。

3.2 加入冷醋酸酐溶液：向沉淀中加入5%冷醋酸酐溶液，使蛋白质沉淀更加纯净。

3.3 混匀：轻轻地旋转样品管或反复倒置，使沉淀和冷醋酸酐溶液充分混合。

3.4 离心：使用高速离心机将样品离心10分钟，以去除醋酸酐和杂质。

四、蛋白质溶解4.1 加入蛋白质溶解液：向蛋白质沉淀中加入适量的蛋白质溶解液，如SDS-PAGE样品缓冲液。

4.2 混匀：轻轻地旋转样品管或反复倒置，使蛋白质沉淀溶解。

4.3 离心：使用高速离心机将样品离心5分钟，以去除残留的杂质。

4.4 收集上清液：将上清液转移到新的离心管中，以便后续的实验使用。

五、存储和分析5.1 存储：将蛋白质溶液存储在-20℃的冷冻离心管中，避免蛋白质的降解和失活。

5.2 浓度测定：使用合适的蛋白质浓度测定方法（如Bradford法），确定蛋白质的浓度。

5.3 SDS-PAGE分析：将蛋白质溶液进行SDS-PAGE电泳，以确定蛋白质的分子量和纯度。