Home | Achievement | Programmes | Projects | Experts | Staffs | Publications | Journals |
Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
MOLECULAR BIOLOGY: WORKING WITH PROTEINS
PURIFICATION: EXTRACT FROM CELLS
This protocol provides considerations and generalized procedures for the use of dialysis to alter the composition of a protein solution. It is a useful step in adjusting a protein sample from one buffer to another, in adjusting the metal and salt ion concentrations, and in the removal of unwanted small molecules. Procedure
A. Preparation of Dialysis Tubing
Choice of Tubing:
1. Dialysis tubing is essentially a cylindrical membrane containing pores. The size of these pores determines the molecular weight "cut-off" of the tubing. Typical cut-offs are 5,000 Daltons, 10,000 Daltons, 30,000 Daltons, and 100,000 Daltons. Thus, if you choose tubing with a cut-off of 30,000 Daltons, all molecules smaller than 30 kD will be able to pass freely through the membrane (see Hint #1).
2. Dialysis tubing can be purchased in two forms: one arrives as a "dry" roll in a box while the other is wet and stored in a liquid buffer. The dry roll must be pre-treated before use. This involves soaking the membrane in ddH2O, heating the membrane to 60°C in Bicarbonate Solution, and rinsing in ddH2O. The wet membrane has already been pre-treated for use and only needs to be rinsed in ddH2O to remove preservatives (see Hint #2).
B. Use of the Dialysis Tubing
1. After washing the tubing (see Protocol ID#113), calculate the length of tubing necessary to contain the volume of your protein sample and cut the tubing to length, leaving an extra inch or two on each side for closures (see Hint #3).
2. Use a dialysis tubing-specific closure (e.g., Spectra/Por Closures from Spectrum Laboratories) to close one end, or tie two tight knots on one end of the tubing (see Hint #4).
3. Pipette your protein sample into the tubing and close off the other end of the tubing with another closure or with two tight knots. Try to avoid including too many air bubbles (see Hint #5).
4. Insert the dialysis tubing containing your protein into Dialysis Buffer in a large beaker. The volume of the buffer should be at least 100 times the original volume of the protein sample. The buffer should also be pre-chilled if your protein is labile.
5. Stir the buffer slowly with a stirbar and magnetic stir plate for at least 1.5 hr.
6. Discard the Dialysis Buffer and replace with the same volume of fresh Dialysis Buffer.
7. Dialyze for an additional 1.5 hr (see Hint #6).
8. Remove the tubing from the beaker containing Dialysis Buffer and carefully open one end of the tubing. Pipette the dialyzed protein solution into tubes.
Bicarbonate Solution 2% (w/v) NaH2CO3
1 mM EDTA
Dialysis Buffer Metal and Salt Ions (NaCl, KCl, MgCl2, etc.)
Other small molecules (Glycerol, DTT, ATP, etc.)
Typical Protein Buffering Agent (Tris, HEPES, Phosphate-based, etc.)
BioReagents and Chemicals
1. Choose your cut-off wisely (below the size of your protein), especially if you have spent several days in the coldroom purifying your protein and are using dialysis as a last purification step. Otherwise, you will quickly discover that your protein has disappeared.
2. The contributor of this protocol suggests using the wet dialysis tubing. However, as the wet tubing is usually more expensive than the dry roll, the Principal Investigator should be consulted.
3. Avoid the introduction of contaminants by wearing gloves when handling the tubing; otherwise, you may discover that your protein has disappeared or that you have purified human keratin. Tubing can be purchased in a variety of diameters (or widths). The contributor of this protocol suggests that you impress your colleagues by "eyeballing" the sufficient length. Avoid any attempt to impress your Principal Investigator by asking for assistance in this calculation.
4. You can fill the tubing with a small amount of ddH2O and squeeze the tubing from the open end to make sure the closure is tight.
5. Create a small protein dialysis "sausage" when closing off the second end of the tubing. BE CAREFUL, however, to leave enough room for expansion, especially if your protein sample contains high salt concentrations and you are dialyzing against a buffer that contains low salt concentrations. Otherwise, you may discover that your protein has disappeared and your sausage has burst. Bubbles smaller than approximately 250 μl are fine.
6. The exchange of solution components between the protein sample and the Dialysis Buffer is most efficient if carried out for longer than 1.5 hr. This should be empirically determined. However, an overnight incubation is often optimal for removal of small molecules from a protein sample.
Citation and/or Web Resources
1. Spectrum Laboratories contains additional useful information.