沉淀蛋白质的常用方法(TCA、乙醇、丙酮沉淀蛋白操作步骤) (2)

沉淀蛋白质的常用方法（

T

C

< br>A

、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, dete

rgent).

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 (prepar

ation 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 su

pernatant 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 (aceton

e 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 sam

ples in a minimal volume of sample buffer. (The presence of some TCA can give a yel

low colour as a consequence of the acidification of the sample buffer titrate with 1N Na

OH 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% f

inal concentration. Mix and keep 5min

–

20oC and then 15min 4oC; or longer time at 4o

C without the

–

20oCstep for lower protein concentration. Suggestion:

leave ON if the protein concentration is very low. (preparation of 100% TCA: 454ml H

2O/kg TCA. Maintain in dark bottleat careful, use gloves!!!).

2) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge su

pernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be dif

ficult to see).

3) For PAGE-SDS, resuspend samples in a minimal volume of sample buffer. (The pr

esence 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 blu

e 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 le

ast 20min

–

20oC. (Suggestion: leave ON if the protein concentration is very low).

2) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge su

pernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be dif

ficult 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

befo

re PAGE-SDS

1) Add to 1 volume of protein solution 9 volumes of cold Ethanol 100%. Mix and kee

p at least

–

20oC. (Suggestion: leave ON).

2) Spin 15min 4oC in microcentrifuge at maximum speed (15000g). Carefully discha

rge 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 5

min 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 buffe

r.