SDS PAGE protein gels
26 Oct 2014Resources:
Pouring your own SDS-PAGE gel
- Janet will handle this for the forseeable future.
Pre-run SDS-PAGE gel if hand-poured
- There is some sort of salt front that messes up low MW bands in our hand-poured gels, but pre-running the gel for 30 minutes solves the problem.
- Load the gel with fresh or fresh-ish 1X buffer, and run for ~30 min at 200V to solve the problem.
Preparing samples
- Decide how much sample to load per well
- Often it is useful to load a few different concentrations so you are likely to get a loading that is informative (not under or over loaded).
| protein source | amt. to load | comments |
|---|---|---|
| total cell protein (lysed & spun down | 5-8 or even 20ug per well (mini protean 10-well comb) | |
| cell cultures (total protein) | ~5-8 uL of turbid TB E. coli for skinny wells. Becomes 10uL after adding 4X buffer. Or, 2uL*OD is appropriate (NanoDrop 10mm OD units). | |
| purified protein | 4-8 uL*uM | works well for ~50-70kDa protein. |
| purified protein | ~40ng | So minimum concentration is ~5ng/uL. If stock is Good for ~50-70kDa proteins. |
Decide how much excess to prepare
- Planning what you will load each lane in advance is helpful for this.
- If the protein isn’t precious, make a few times more than is required so you can re-run if necessary.
Prepare samples
- Use Janet’s home-made 4X loading dye. Before use, add 4% by volume beta mercaptoethanol (b-ME) so the final sample will have 1%.
- Prepare a little excess (~10%)
- Add loading dye to samples in PCR tubes
- Boil for 5 min on a thermocycler
- 100oC for 5 minutes
Run gel
- Janet has been pre-running the gel for 15 min at 60V, but I’m not convinced this is necessary.
- Run at 200V for ~30 min, check gel, then keep running until the band you want to detect is near the bottom of the gel.
- ACS is ~ 74.5 kDa
- ADH 3K9D is ~ 49 kDa
- The ladder we usually use is PageRuler Prestained.
Wash & Stain gel
- Wash SDS (soap) out of the gel by taking it out of the plates, and putting it in a tupperware of fresh DI water.
- Let stand ~10 min for equilibrium. Repeat 3 times. Insufficient washing –> whole gel stains & you can’t see where the bands are.