2024年5月9日发(作者：苹果iphone已停用连接itunes怎么才能解开)

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

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

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 –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 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.