PROTEIN PRECIPITATIONS (TCA, METHANOL, AMMONIUM SULPHATE) |
|
Last Update: December 2006 |
|
|
|
| PREPARE SOLUTIONS | |
| 1. 100% TCA (500 mL): | Mix 227 mL of dH2O with 500 g of TCA crystals (keep in the dark) |
| PROCEDURE - TCA precipitation | |
| 1. To 100 mL of protein sample, add 11 mL of 100% ice-cold TCA (to a final concentration of 10%) | |
| 2. Mix and keep on ice for >45 minutes to 3 hrs (cold room) | |
| 3. Centrifuge at 4oC for 30-45 minutes | |
4. Remove supernatant and add 0.9-1.5mL of 100% acetone to wash pellet |
|
| 5. Vortex throughly | |
| 6. Centrifuge at 4oC for 30-45 minutes | |
| 7. Remove supernatant | |
| 8. Repeat steps 5-7 one more time | |
9. Dry doe 1 minute in SpeedVac |
|
| 10. Resuspend in 1 vol 1X SDS-PAGE Loading buffer 1/10 vol of 1M Tris pH 8.0 (or until color turns blue) | |
| NOTE: Residual acid has to be buffered with Tris otherwise the protein won't resuspend | |
| 11. Vortex well | |
12. Store at 4oC |
|
| PROCEDURE - Methanol/Chloroform protein extraction | |
| Note: very useful to remove detergents (Triton X-100) from protein samples. Specially useful for membrane protein preparations | |
| 1. Mix 100 mL of sample, 400 mL of methanol, 100 mL of chloroform, and 300 mL of dH2O (all ice cold), vortex throughly and place on ice for >2 minutes | |
| 2. Spin for >2 minutes at 10,000 rpms | |
| 3. Remove supernantant (leaving protein interphase) | |
| 4. Add 300 mL of methanol, vortex throughly and spin for >5 mins at 10,000 rpms | |
| 5. Remove supernatant and resuspend pellet in favorite buffer | |
| PROCEDURE - Ammonium sulphate precipitation of proteins | |
1. To the sample, add a determined amount of saturated ammonium sulfate solution such that the final concetration is 10% (depending on the proteins of interest, ammonium sulfate concentrations form 10 to 90% can de tried). Add dropwise to prevent local precipitation of proteins |
|
| NOTE: solid ammonium sulfate can also be added instead of using a saturated solution | |
2. Vortex as the ammonium sulfate solution is added as well as the the end. Leave on ice for >1 hour |
|
3. Spin at 10,000 rpms for >5 minutes |
|
| 4. Remove supernatant and resuspend in favorite buffer | |