"Low pH Cleavage Buffer" Preparation
Updated 6/12/2015 08:33 PM
Introduction
Materials
- Consumables
- phosphate buffered saline (PBS) (concentrated)
- Bis-Tris
- EDTA
- hydrogen chloride (HCl) (concentrated)
- Disposables
- pipette tips
- Equipment
- pipette
- sterile container
- pH meter
Procedure
Components
Table1
A A | B B | |
1 1 | Final Volume (mL)* | 100 |
Table2
A A | B B | C C | D D | |
1 1 | Component | Stock Concentrate (x)* | Stock Concentration (M)* | Volume (mL) |
2 2 | PBS (concentrate) | 10 | 10 | |
3 3 | bis-tris | 1 | 4 | |
4 4 | EDTA | 0.5 | 0.4 | |
5 5 | dH2O | 85.6 |
The dH2O component is used to calculate the remaining volume after components have all been added. If an input is empty, it is unused for that component.
Procedure
- Add ~80% of the final volume of dH2O to a sterile container.
Generally, the capacity of the container should be greater than (not equal to) the final volume of solution to be prepared in order to prevent the solution from boiling over during the autoclave step.
Ensure you have a volumetric flask measuring your desired final volume.
- Adjust the final volume and stock concentrate values in the table above and add the calculated amounts of the components to the dH2O.
- Titrate solution to pH 6.2 using concentrated HCl.
- Once completely dissolved, transfer the solution to a sterile volumetric flask and adjust the final volume using dH2O, then return it to the container.
Ensure the solution is well mixed before returning it to the container.
- Apply autoclave tape to the bottle and autoclave on liquids cycle.
- Store at room temperature.
This protocol describes the preparation of the cleavage buffer used in the dual ELP-tagged split-intein system of protein expression and purification.
Fong, B. A., Wu, W.-Y. & Wood, D. W. (2009). Optimization of ELP-intein mediated protein purification by salt substitution. Protein Expr Purif 66, 198-202.
Shi, C., Meng, Q. & Wood, D. (2013). A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification. Appl Microbiol Biotechnol 97, 829-835.