Changing buffers for your GST.
1. Dialysis
Tubing with pores that have size smaller than the protein but larger than the
buffer contents.
Advantages
Gentle on the protein
Inexpensive
Can be done in cold room
Not labor intensive
Better for larger volumes
Disadvantages
Takes time. Couple days is best for complete buffer change
Risk of losing sample while handling tubing
Sample difficult to recover if lost through tubing or clips
2. Desalting Column
Use size exclusion resin. Sephadex G25, or pre-packed PD-10 columns
Advantages: Quick
Disadvantages: incomplete desalting
Not best for changing to a new buffer
More expensive
Dilutes protein
3. Spin Column
Attach a micro column to an eppi tube
Place sample in column and spin
Buffer passes through membrane, protein is retained in column cup
Advantages
More complete than desalting if repeated serially
Relatively fast
Can also concentrate the protein at the same time
Sample easily recovered if leakage occurs
Disadvantages
Limited volumes can be done at a time
Expense
Serial spins may require some time depending on cutoff size
Risk of drying sample, risk of precipitation of protein
Buffer inside
beads
GST passes around
beads and elutes
first without salts
