Protein precipitation
Reagents:
- Trichloroacetic acid from Sigma, #T9159. Prepare 50% solution, store at RT. Cool on ice before use.
- Acetone from Sigma, #179124. Store at -20C.
- Insulin (if necessary) from Sigma, #I-5500.
- 1 X SDS-PAGE buffer (no dye): 50 mM Tris, 2% SDS, 10% glycerol, 100 mM DTT, pH 8.0.
Protein precipitation may be used to concentrate protein extracts, sample fractions from chromatography, fractions form sucrose gradient analysis, etc for various downstream use.
1. If a small amount of protein is to be concentrated, some insulin (~ 10 ug) may be added as a carrier.
2. Add 1/2 volume of ice-cold 50% TCA into samples, and vortex. Be extremely careful about the TCA since it may dissolve your finger as well.
3. Keep on ice for 30 min.
4. Spin down at 14k RPM for 10 min at 4C.
5. Transfer the samples to ice. A transparent or white pellet may be seen. Occasionally, the pellet is very small and transparent and it it hard to find.
Just proceed to the next steps.
6. Pour the supernatant. Be careful not to accidentally lose the pellet. Again, you may not see the pellet.
7. Add 300 ul cold acetone. Gently flick the side of the tube to dissolve pellet in acetone. The protein will be forming small clumps while most of other
materials will be completely dissolved. However, do not work on one sample for too long. Put back on ice every 2-3 min until the pellet is gone.
8. Spin down at 14K RPM for 10 min at 4C.
9. Pour the supernatant. Be careful not to accidentally lose the pellet.
10. Open the tubes and air dry in the fume hood for 5-10 min. Do not overdry your sample since it is hard to be dissolved again.
11. You may dissolve the pellets with distilled water or any other solvents. I usually use 1 X SDS-PAGE buffer (without dye). Depending on the total protein,
I typically use 20 ul to dissolve the pellet overnight at 4C. Or you may put the tube to 65C shaker for 30 min.
12. Add 5 ul 2 X SDS-PAGE buffer (with dye and DTT) to each sample. The leftover of TCA and acetone may acidify the solution. If you see the sample becomes
yellow, you may add 5 ul 1 M Tris, pH 9.0 to addjust it back to alkaline range.
13. Boil the sample. Ready to go for regular SDS-PAGE.