沉淀蛋白质的常用方法

沉淀蛋白质的常用方法（TCA、乙醇、丙酮沉淀蛋白操作步骤）TCA-DOCFor precipitation of very low protein concentration1) 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 to obtain the normal blue sample buffer colour.)Normal TCATo eliminate TCA soluble interferences and protein concentration1) 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 ofthe acidification of the sample buffer ; titrate with 1N NaOH or 1M TrisHCl to obtain the normal blue sample buffer colour.)Acetone PrecipitationTo eliminate acetone soluble interferences and protein concentration1) 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 PrecipitationUseful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS1) 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/AcetoneUseful method to concentrate proteins and remove acetone and TCA soluble interferences1. To one volume of protein solution add 2% Na deoxycholate (DOC) to % 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 to obtain the normal blue sample buffer colour.)Acidified Acetone/MethanolUseful method to remove acetone and methanol soluble interferences like SDS before IEF1) 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 PrecipitationUseful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS1) Dilute 10-25μl samples to 100μl with HO2Add 100μl of 20% trichloroacetic ac id (TCA) and mix (preparation of 100% TCA: 454ml HO/kg TCA. Maintain in dark bottleat careful, use2gloves!!!).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°C6) (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 to obtain the normal blue sample buffer colour.)PAGE prep TM Protein Clean-up and Enrichment Kit - PIERCEThe 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 PrecipitationUseful method for Removal of salt and detergents1) To sample of starting volume 100 ul2) Add 400 ul methanol3) Vortex well4) Add 100 ul chloroform5) Vortex6) Add 300 ul H2O7) Vortex8) Spin 1 minute @ 14,0000 g9) Remove top aqueous layer (protein is between layers)10) Add 400 ul methanol11) Vortex12) Spin 2 minutes @ 14,000 g13) Remove as much MeOH as possible without disturbing pellet14) Speed-Vac to dryness15) Bring up in 2X sample buffer for PAGEReference: Wessel, D. and Flugge, U. I. Anal. Biochem. (1984) 138, 141-143蛋白质浓缩——方法很全1130徐炉李2011-05-28 14:35楼主蛋白质浓缩——方法很全 - 丁香园论坛-医学/药学/生命科学论坛蛋白质浓缩方法总结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。