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

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

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

–

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.

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 H

2

O

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

454ml H

2

O/kg TCA. Maintain

in dark bottleat 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 prep

TM

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 prep

TM

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