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-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 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/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 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°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 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 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

蛋白质浓缩——方法很全113 0

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，而且透析袋只要不破裂，可以反复使用。

可以用millipore 公司的amicon ultra－15 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，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

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