in progress! very rough.
This procedure can be used either for concentration of protein samples or for buffer exchange.
Just some notes.
- Pour column in a single step using a suspension of no more than 2 times the settled volume so that beads don't separate by size.
- Drain buffer to surface.
- Close clamp.
- Apply sample with pipette while moving the pipette around the column.
- Once sample has drained to surface, close clamp.
- Apply buffer with pipette.
- Once buffer has drained to surface, close clamp.
- Sample should be contained in a small volume.
- Applied sample volume should not exceed 3% of total column volume with 1% being ideal.
- Fractions eluted earlier are less spread than fractions eluted later (because of less time spent on the column). More spreading risks the protein being contaminated by similar sized proteins. But if there are a lot of larger sized unwanted protein around, then early fractions may overlap.
- Usually columns are 20-40 times longer than their diameter.
- Avoid applying a sample that is less dense than the buffer because the follow up buffer will tend to run below the end of the sample thereby causing the last part of sample to enter late.
- The buffer that the protein will elute in is the buffer present in the column before starting it.
- Protein concentration should be 10-20 mg/mL with 30 mg/mL at the most.
- Ideal column size: diameter where m = amount of protein in mg and length = 30 * diameter.
- Gel filtration: Principles and Methods by Amersham Pharmacia Biotech pdf