I now work in a lab with a protein and a pre-made purification protocol, were they purify a His-tagged protein using linear elution which then gives a steady increase in the imidazole concentration. However, I am curious to as if you plainly have to test which elution scheme you want to use, or if there are some "rule-of thumb" on what is a more standard way to elute protein, and what in general is more common?
Also, say for instance (just as an example) that I use a protocol which in the end loses about 80% of the protein before it's pure, is one of the first things you could do is to change the elution schemes to increase the yield? Are there other very basic things one can do to optimize a purification protocol which has not been so thoroughly optimized?
- Is linear "usually" better/more common than stepwise?
- Is imidazole gradient "usually" better/more common than pH gradient?