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-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)
2、 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 i
3、nversion 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 PA
4、GE-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.)
5、 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 –20oCst
6、ep 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). Caref
7、ully 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 o
8、f the acidification of the sample buffer titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.) Acetone Pr
