Elution buffer
| Reagent | Final concentration | Amount |
|---|---|---|
| L-glycine (molar mass: 75.07 g/mol) | 0.64 M | 4.85 g |
| Urea (molar mass: 60.056 g/mol) | 3 M | 18.02 g |
| Guanidinum chloride (molar mass: 95.53 g/mol) | 3 M | 28.66 g |
| TCEP (molar mass: 286.65 g/mol) | 70 mM | 2 g |
| 3 N HCl | (to pH 2.5) | X mL |
| ddH2O | complete volume to 100 mL | |
| Total | 100 mL |
Note: Dissolve glycine, urea, and guanidinium chloride in water first before adding TCEP. After the addition of TCEP, avoid incorporating air into the buffer by stirring slowly. Adjust pH of the solution to 2.5 with HCl and add ddH2O to the final volume. “X” denotes the volume of 3 N HCl needed to bring the solution to pH 2.5. In our experience, 12–14.5 mL of 3 N HCl is typically sufficient to bring the pH to 2.5. Filter with 5-micron syringe filter. Store at 4°C for <1 month. TCEP can degrade and become ineffective over time.