沉淀蛋白质的通用方法(TCA,乙醇,丙酮沉淀蛋白操作技巧步骤)

,.

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

μ

l sample, add 1

μ

l 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

μ

l 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 + 10

μ

l 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 10-25

μ

l samples to 100

μ

l with H

2

O

Add 100

μ

l 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 100

μ

l 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

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

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蛋白质浓缩方法总结

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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