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 111,1ml TCA)
2.Incubate 30 min on ice.
3.Spin in microfuge at 4°C for 15 min.
4.Carefully remove all supernatant. Dry drops at the inner wall of the Eppi with a piece of Kleenex or Kimwipes without touching the pellet.
5.Add 300 ml cold acetone and spin 5 min at 4°C.
Optional:
6.Remove supernatant and dry pellet.
7.Resuspend samples in SDS PAGE loading buffer. Load to SDS PAGE after heating at 65°C for 3 min. For samples with low protein concentration:
Before precipitating with TCA add 1% deoxycholic acid (DOC, Na+ salt) to make your protein solution 0,1% DOC. DOC will precipitate in acidic solution and then co-precipitate the proteins. This will give usually a big, fat pellet, most of which 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 any more. The sample is then lost 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.
