Preparation of samples for SDS-PAGE by precipitation with Trichloroacetic acid (TCA) and alternative methods

1. Add trichloroacetic acid (TCA) to the extract to a final concentration of 10-20 and allow the proteins to precipitate on ice for 30 min.

 

2. Spin the sample at 14 000 rpm for 30 min in a table centrifuge at +4C.

 

3. Remove liquid and wash pellet with precooled (-20C) acetone to remove residual TCA: incubate on ice for 10 min, spin at 14 000 rpm for 10 min, remove acetone.

 

Repeat the washing once again. Remove residual acetone by air drying. Resuspend the sample in 20-30 l of Sample buffer (with -mercaptoethanol), heat at +95C for 1-2 min, and spin for several seconds in a table centrifuge. The sample is ready for the SDS-PAGE.

 

Alternatively:

  • Precipitate the sample with 9 volumes of aceton overnight at -20C. Remove liquid and do not dry the pellet too much! otherwise it will be impossible to resolubilize the proteins. However, this method is better then precipitation with TCA!!!

  • Precipitate with 9 volumes of cold 95 EtOH for 2 hr at -20C, and remove liquid. This method is worse than precipitation with TCA!!!

 

2003 08 15 – Ove, Oliver & Julius