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川普当选
For precipitation of very low protein concentration
1) To one volume of protein solution, add 1/100 vol. of 2% DOC (Na deoxycholate, detergent).
hit的过去式2) V ortex and let sit for 30min at 4oC.
quoted printable3) 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, u 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 e). [OPTION: W ash 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 PA GE-SDS, resuspend samples in a minimal volume of sample buffer. (The prence of some TCA can give a yellow colour as a conquence of th
e 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, u 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 e).
3) For PA GE-SDS, resuspend samples in a minimal volume of sample buffer. (The prence of some TCA can give a yellow colour as a conquence 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 e).
3) Dry samples under vaccum (speed-vac) or dry air to eliminate any acetone residue (smell tubes). For PA GE-SDS, resuspend samples in a minimal volume of sample buffer.
Ethanol Precipitation
Uful method to concentrate proteins and removal of Guanidine Hydrochloride before P AGE-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 e).
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3) Wash pellet with 90% cold ethanol (keep at –20oC). V ortex 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 PA GE-SDS, resuspend samples in a minimal volume of sample buffer.
TCA-DOC/Acetone
Uful method to concentrate proteins and remove acetone and TCA soluble interferences主谓一致
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, u 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 (s mell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.
10. (The prence of some TCA can give a yellow colour as a conquence 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
Uful method to remove acetone and methanol soluble interferences like SDS before IEF
1) Prepare acidif ied acetone: 120ml acetone + 10μl HCl (1mM final concentration).
2) Prepare precipitation reagent: Mix equal volumes of acidified acetone and methanol and keep at -20oC.
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成考自考区别3) To one volume of protein solution add 4 volumes of cold precipitation reagent. Mi x 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 e).
5) Dry samples under vaccum (speed-vac) or dry air to eliminate any acetone or methanol residue (smell tubes).
故弄玄虚英语TCA-Ethanol Precipitation
Uful method to concentrate proteins and removal of Guanidine Hydrochloride before P AGE-SDS 1) Dilute 10-25μl samples to 100μl with H2O
Add 100μl of 20% trichlo roacetic acid (TCA) and mix (preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat 4oC.Be careful, u 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 e).
4) Wash pellet with 100μl ice-cold ethanol, dry and resuspend in sample buffer.
5) In ca there are traces of GuHCl prent, samples should be loaded immediately after boiling for 7 min at 95°C
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6) (The prence of some TCA can give a yellow colour as a conquence 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 - PIERCE
The P AGEprep? Kit enables removal of many chemicals that interfere with SDS-P AGE analysis: guanidine, ammonium sulfate, other common salts, acids and bas, detergents, dyes, DNA, RNA, and lipids.
PIERCE: #26800 - PA GE prep TM Protein Clean-up and Enrichment Kit(pdf)
Chloroform Methanol Precipitation
Uful method for Removal of salt and detergents
2)1) To sample of starting volume 100 ul
2) Add 400 ul methanol
3) V ortex well
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4) Add 100 ul chloroform
5) V ortex
6) Add 300 ul H2O
7) V ortex
8) Spin 1 minute @ 14,0000 g
9) Remove top aqueous layer (protein is between layers)
10) Add 400 ul methanol
11) V ortex
12) Spin 2 minutes @ 14,000 g
13) Remove as much MeOH as possible without disturbing pellet
14) Speed-V ac to dryness
15) Bring up in 2X sample buffer for PA GE
Reference: W esl, D. and Flugge, U. I. Anal. Biochem. (1984) 138, 141-143
三氯乙酸（TCA）沉淀蛋白的原理
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TCA与蛋白质之间主要有以下几个方面的作用：
①在酸性条件下与蛋白质形成不溶性盐；
②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；
③随着蛋白质分子量的增大，其结构复杂性与致密性越大，TCA可能渗入分子内部而使之较难被完全除去，在电泳前样品加热处理时可能使蛋白质结构发生酸水解而形成碎片，而且随时间的延长这一作用愈加明显；
④电泳图谱显示，BSA、HSA单体谱带有较明显的展宽现象，这可能是由于TCA的结合，使SDS 与蛋白质的结合量产生偏差，从而造成蛋白质所带电荷的不均一性，造成迁移率的不一致。
我们认为在电泳时使用TCA对蛋白质样品的浓缩或除盐时，对于分子质量大的蛋白质，要慎重选择TCA。
对小分子量蛋白质的浓缩，采用TCA时也有两点需要注意：一是用TCA沉淀后，尽量用丙酮彻底抽提TCA；二是样品处理后要尽快进行电泳分析，以免发生聚集及断裂，造成结果分析的不准确。
