发布时间 : 星期二 文章沉淀蛋白质的常用方法（TCA、乙醇、丙酮沉淀蛋白操作步骤）更新完毕开始阅读b14ae287482fb4daa58d4bb1
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 4oC.Be 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 4oC.Be 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 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 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 4oC.Be 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 H2O
Add 100μl of 20% trichloroacetic acid (TCA) and mix (preparation of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat 4oC.Be 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 prepTM 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 prepTM 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