Dr. Mario Lebendiker

Protein Purification Unit

TCA-DOC

For very low protein concentration

To one volume of protein solution, add 1/100 vol. of 2% DOC (deoxycholate).

Vortex and let sit for 30min at 4C.

Add 1/10 of TCA 100% vortex and let sit ON at 4C. (preparation of 100% TCA: 454ml H2O/kg TCA. Maintain 4C in dark bottle).

Spin 10min 4C in microfuge at maximum rpm (15000g). Carefully discharge supernatant and retain the pellet. Dry tube by inversion on tissue (pellet may be difficult to see).

Wash pellet 2x with one volume cold acetone (acetone keep at -20C). Vortex and repellet samples 5min at full speed between washes.

Dry samples under vaccum (speed vac) or dry air.

Normal TCA

Add to the protein solution TCA to 13% final concentration. Mix and keep 5min -20C and 15min 4C; or longer time at 4C without the -20C step.

Spin 15min 4C in microfuge at maximum rpm (15000g). Carefully discharge supernatant and retain the pellet. Dry tube by inversion on tissue (pellet may be difficult to see).

Acetone Precipitation

Add to 1 volume of protein solution 4 volumes of cold acetone. Mix and keep at least 20min. at -20C. (Suggestion: leave ON).

Spin 10min 4C in microfuge at maximum rpm (15000g). Carefully discharge supernatant and retain the pellet. Dry tube by inversion on tissue (pellet may be difficult to see).

Wash pellet with one volume cold acetone (acetone keep at -20C). Vortex and repellet samples 5min at full speed between washes.

Dry samples under vaccum (speed vac) or dry air.

Ethanol Precipitation

Preparation of samples containing Guanidine Hydrochloride for PAGE-SDS

Add to 1 volume of protein solution 9 volumes of cold Ethanol 100%. Mix and keep at least 10min. at -20C. (Suggestion: leave ON).

Spin 10min 4C in microfuge at maximum rpm (15000g). Carefully discharge supernatant and retain the pellet. Dry tube by inversion on tissue (pellet may be difficult to see).

Wash pellet with 90% cold ethanol (keep at -20C). Vortex and repellet samples 5min at full speed.

Dry samples under vaccum (speed vac) or dry air. Resuspend samples in sample buffer.