Quantitative Staining

Quantitative Staining
1. Add 0.5 g Coomassie Brilliant Blue G-250 to approx. 10 ml RO H2O in a 15 ml centrifuge tube.
Stored in Room Temperature Reagent Desiccator.
2. Vortex and place on rocking platform.
3. Add 400 ml of Stock Staining solution to 500 ml graduated cylinder.
4. Add 100 ml methanol to cylinder for a total of 500 ml.
The solution will appear milky initially then turn to clear as the solution is mixed as it is poured into the staining tray.
Methanol is TOXIC and is absorbed through unprotected skin.
Use gloves and eye protection.
5. Incubate gel in TCA solution for 60 min on rocking platform.
6. Hold gels to bottom of staining tray with one hand and invert to discard TCA solution into sink.
7. Rinse with two changes of DI H2O.
8. Add Stock-Staining-Solution/Methanol from cylinder.
9. Add Coomassie staining solution, which will include particulate material.
10. Stain 8 hrs (or overnight) on rocking table.
11. Add 100 ml of methanol to 500 ml graduated cylinder.
12. Add 300 ml of RO H2O to cylinder to a total of 400 ml (25% methanol).
13. Remove staining solution by imversion
14. Wash any remaining stain particles off the sides of the staining tray with the methanol squeeze bottle.
15. Add 25% methanol de-staining solution.
16. De-stain for 4 hrs.
17. Replace with fresh 25% methanol solution and de-stained for an additional 4 hrs.
18. Remove de-staining solution, rinse gels twice with DI H2O
19. Store in 25% ammonium sulfate solution.