Protein Precipitation Protocols
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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.)
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.)
