Methods:Methanol:Chloroform Protein Precipitation
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Removes SDS, TX-100, salts, beta-mercaptoethanol, etc.
PROCEDURE: To 100 ul of sample:
1. Add 400 ul of MeOH; vortex
2. Add 100 ul of Chloroform; vortex
3. Add 300 ul MilliQ water; vortex
4. Spin at high speed in microfuge
5. Result: A layer at interface (= protein)
6. Remove and discard the upper layer
7. Add 300 ul MeOH; vortex
8. Spin at high speed in microfuge
9. Proteins in pellet
10. Remove supernatant materials
11. Wash pellet with MeOH
12. Remove MeOH
13. Air dry the pellet
14. Resuspend pellet in buffer of choice
- Taken from Chapter 1 of the DIGE Training Manual
This procedure can be adapted to larger volumes. For example, 1 ml sample is precipitated by adding 4 ml MeOH, 1 ml chloroform and 3 ml water. After centrifugation and removing the upper layer, the lower layer and interface proteins are transferred to 1.5 ml eppendorf tubes and the procedure is applied as described above. (The ratio of protein:MeOH: chloroform: water is 1:4:1:3).
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