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1、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 at4oC (prepar
2、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 supernatant and retain the pellet: dry tube by inversion ontissue paper (pellet may be difficultto see).OPTION: Washpellet twicewith one
3、volume of cold acetone (acetone keep at 20oC). Vortex andrepellet 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 TCAcan give a yellow colour as a consequence of t
4、he acidification of thesample 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. Mi
5、x and keep 5min 20oC and then 15min4oC; or longer time at 4oC without the20oC step for lower proteinconcentration.Suggestion:leave ONifthe proteinconcentrationisverylow.(preparation of 100% TCA: 454ml H2O/kg TCA. Maintainin dark bottleatcareful, use gloves!).2) Spin 15min 4oC in microfuge at maximum
6、 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 acidificati
7、onof the sample buffer; titratewith 1N NaOHor 1M TrisHClto 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. keep at least 20min 20oC. (Suggestion: le
8、ave ON if the proteinMix andconcentration 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 elimina
9、te 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 kee
10、p at least 20oC. (Suggestion: leave ON).2) Spin 15min 4oC in microcentrifugeat maximumspeed (15000g).Carefullydischarge supernatant and retain the pellet: dry tube by inversion ontissue paper (pellet may be difficult to see).3) Wash pelletwith 90% cold ethanol (keep at20oC). Vortex andrepellet sampl
11、es 5min at full speed.4) Dry samples under vaccum (speed vac) or dry airto eliminateany ethanolresidue (smelltubes).For PAGE-SDS,resuspend samples in a minimal volumeof sample buffer.TCA-DOC/AcetoneUseful method to concentrate proteins and remove acetone and TCAsoluble interferences1. To one volume
12、of protein solution add 2% Na deoxycholate (DOC) to %final (for 100l sample, add 1l 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 glov
13、es!).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 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 microcentrifu
14、ge 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 buff
15、er ; 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 + 10 l HCl (1mM finalconcentration).2)Prepare precipitationreagent:Mix equal volumes of acidified acetoneand methanol and keep at -20oC.3)
