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 15:19

TCA-DOC

For precipitation of very low protein concentration

1) 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 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 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 Precipitation

To eliminate acetone soluble interferences and protein concentration

1) 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 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 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 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 + 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 Precipitation

Useful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS

1) Dilute 10-25μl samples to 100μl with H2O

Add 100μl of 20% trichloroacetic acid (TCA) and mix (preparation 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°C

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 - PIERCE

The 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 Precipitation

Useful method for Removal of salt and detergents

1) To sample of starting volume 100 ul

2) Add 400 ul methanol

3) Vortex well

4) Add 100 ul chloroform

5) Vortex

6) Add 300 ul H2O

7) Vortex

8) Spin 1 minute @ 14,0000 g

9) Remove top aqueous layer (protein is between layers)

10) Add 400 ul methanol

11) Vortex

12) Spin 2 minutes @ 14,000 g

13) Remove as much MeOH as possible without disturbing pellet

14) Speed-Vac to dryness

15) Bring up in 2X sample buffer for PAGE

Reference: 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。在浓缩胶中，其pH环境呈弱酸性，因此甘氨酸解离很少，其在电场的作用下，泳动效率低；而CL离子却很高，两者之间形成导电性较低的区带，蛋白分子就介于二者之间泳动。由于导电性与电场强度成反比，这一区带便形成了较高的电压剃度，压着蛋白质分子聚集到一起，浓缩为一狭窄的区带。当样品进入分离胶后，由于胶中pH的增加，呈碱性，甘氨酸大量解离，泳动速率增加，直接紧随氯离子之后，同时由于分离胶孔径的缩小，在电场的作用下，蛋白分子根据其固有的带电性和分子大小进行分离。

所以，pH对整个反应体系的影响是至关重要的，实验中在排除其他因素之后仍不能很好解决问题的情况，应首要考虑该因素。

2. 样品如何处理？

根据样品分离目的不同，主要有三种处理方法：还原SDS处理、非还原SDS处理、带有烷基化作用的还原SDS处理。

1）还原SDS处理：在上样buffer中加入SDS和DTT（或Beta巯基乙醇）后，蛋白质构象被解离，电荷被中和，形成SDS与蛋白相结合的分子，在电泳中，只根据分子量来分离。一般电泳均按这种方式处理，样品稀释适当浓度，加入上样Buffer，离心，沸水煮5min，再离心加样。

2）带有烷基化作用的还原SDS处理：碘乙酸胺的烷基化作用可以很好的并经久牢固的保护SH基团，得到较窄的谱带；另碘乙酸胺可捕集过量的DTT，而防止银染时的纹理现象。100ul样品缓冲液中10ul 20％的碘乙酸胺，并在室温保温30min。

3）非还原SDS处理：生理体液、血清、尿素等样品，一般只用1％SDS沸水中煮3min，未加还原剂，因而蛋白折叠未被破坏，不可作为测定分子量来使用。

3. SDS-PAGE电泳凝胶中各主要成分的作用？

聚丙烯酰胺的作用：丙烯酰胺与为蛋白质电泳提供载体，其凝固的好坏直接关系到电泳成功与否，与促凝剂及环境密切相关；

制胶缓冲液：浓缩胶选择pH6.7，分离胶选择pH8.9，选择tris－HCL系统，TEMED与AP：AP提供自由基，TEMED是催化剂，催化自由基引起的聚合反应进行；十二烷基硫酸钠（SDS）：阳离子去污剂，作用有四：去蛋白质电荷、解离蛋白质之间的氢键、取消蛋白分子内的疏水作用、去多肽折叠。

4. 提高SDS-PAGE电泳分辨率的途径？

聚丙烯酰胺的充分聚合，可提高凝胶的分辨率。建议做法：待凝胶在室温凝固后，可在室温下放置一段时间使用。忌即配即用或4度冰箱放置，前者易导致凝固不充分，后者可导致SDS结晶。一般凝胶可在室温下保存4天，SDS可水解聚丙烯酰胺。

一般常用的有氨基黑、考马斯亮蓝、银染色三种染料，不同染料又各自不同的染色方法，具体可参照郭尧君编著的《蛋白质电泳技术手册》P82-103。

5.“ 微笑”（两边翘起中间凹下）形带原因？

主要是由于凝胶的中间部分凝固不均匀所致，多出现于较厚的凝胶中。 处理办法：待其充分凝固再作后续实验。

6. “皱眉”（两边向下中间鼓起）形带原因？

主要出现在蛋白质垂直电泳槽中，一般是两板之间的底部间隙气泡未排除干净。

处理办法：可在两板间加入适量缓冲液，以排除气泡。

7. 为什么带出现拖尾现象？

主要是样品融解效果不佳或分离胶浓度过大引起的。

处理办法：加样前离心；选择适当的样品缓冲液，加适量样品促溶剂；电泳缓冲液时间过长，重新配制；降低凝胶浓度。

8. 为什么带出现纹理现象？

主要是样品不溶性颗粒引起的。

处理办法：加样前离心；加适量样品促溶剂。

9. 什么是“鬼带”，如何处理？

“鬼带”就是在跑大分子构象复杂的蛋白质分子时，常会出现在泳道顶端（有时在浓缩胶中）的一些大分子未知条带或加样孔底部有沉淀，主要由于还原剂在加热的过程中被氧化而失去活性，致使原来被解离的蛋白质分子重新折叠结合和亚基重新缔合，聚合成大分子，其分子量要比目标条带大，有时不能进入分

离胶。但它却于目标条带有相同的免疫学活性，在WB反应中可见其能与目标条带对应的抗体作用。

处理办法：在加热煮沸后，再添加适量的DTT或Beta巯基乙醇，以补充不足的还原剂；或可加适量EDTA来阻止还原剂的氧化。

10. 为什么溴酚蓝不能起到指示作用？

我们在实验中常会遇到溴酚蓝已跑出板底，但蛋白质却还未跑下来的现象。主要与缓冲液和分离胶的浓度有关。

处理办法：更换正确pH值的Buffer；降低分离胶的浓度。

11. 为什么电泳的条带很粗？

电泳中条带很粗是常见的事，主要是未浓缩好的原因。

处理办法：适当增加浓缩胶的长度；保证浓缩胶贮液的pH正确（6.7）；适当降低电压；

12. 为什么电泳电压很高而电流却很低呢？

这种现象一般初学者易出现。比如电压50v以上，可电流却在5mA以下。主要是由于电泳槽没有正确装配，电流未形成通路。包括：a.内外槽装反；b.外槽液过少；c.电泳槽底部的绝缘体未去掉（比如倒胶用的橡胶皮）。

处理办法：电泳槽正确装配即可。

13. 浓缩胶与分离胶断裂、板间有气泡对电泳有影响吗？

这主要出现在初学者中，一般对电泳不会有太大的影响。前者主要原因是拔梳子用力不均匀或过猛所致；后者是由于在解除制胶的夹子后，板未压紧而致空气进入引起的。

14. 凝胶时间不对，或慢或快，怎么回事？

通常胶在30MIN-1H内凝。如果凝的太慢，可能是TEMED，APS剂量不够或者失效。APS应该现配现用，TEMED不稳定，易被氧化成黄色。 如果凝的太快，可能是APS和TEMED用量过多，此时胶太硬易裂，电泳时易烧胶。

15. 电泳时间比正常要长？

可能由于凝胶缓冲系统和电级缓冲系统地PH选择错误，即缓冲系统地PH和被分离物质的等电点差别太小，或缓冲系统的离子强度太高。