To Concentrate Proteins for analysis by SDS PAGE 1.

Add one tenth of the sample volume of 77% TCA (trichloroacetic acid) to your protein sample. (e.g. to 900ml sample add 100ml TCA or to 1000ml add 111,1m l TCA) 2. Incubate 30 min on ice. 3. Spin in microfuge at 4C for 15 min. 4. Carefully remove all supernatant. Dry drops at the inner wall of the Epp i with a piece of Kleenex or Kimwipes without touching the pellet. 5. Add 300 ml cold acetone and spin 5 min at 4C. Optional: 6. Remove supernatant and dry pellet. 7. Resuspend samples in SDS PAGE loading buffer. Load to SDS PAGE after hea ting at 65C for 3 min. For samples with low protein concentration: Before precipitating with TCA add 1% deoxycholic acid (DOC, Na+ salt) to make yo ur protein solution 0,1% DOC. DOC will precipitate in acidic solution and then c o-precipitate the proteins. This will give usually a big, fat pellet, most of wh ich is DOC. To prepare 77% TCA: To make a 77% TCA solution you need to use a balance: weigh in 7,7g TCA and add water to 10g. NOTE: Some proteins (perhaps its the composition of the sample) don't precipitate with TCA; but after adding TCA you can't recycle them anymore. The sample is then lo st and you need to prepare a new one. Always wear gloves and your lab coat when working with TCA! TCA injures the skin and also destroys your clothes.