1) Load 20 to 35 μl of your samples unless you know for a fact that this is too much.
2) In the first lane load 25 μl of positive bloting control. This is lysate stimulated with PMA or LPA. This is good for most CCL39 blots.
3) Molecular Wt Markers - There are now two kinds. One is the BioRad Prestained standards we've always used. The other is MagicMarker. This is a non-stained protein that has an IgG recognition site. That means each of the proteins will bind secondary antibody and will be visible on the film. To use this:
- In the second lane load 10-15 μl of the BioRad standard
- In the SAME well/lane add 3 μl of the MagicMarker standard (found in a box in the
freezer in the main room
4) Run the gel to the bottom but not off. Make certain the dye is just coming to the edge of the glass not the gasket. Remember, 200 v for 1 hour is an approximation - you may very likely have to turn it up a little more. If this is a key experiment, run the gel at 100 v for 10-15 min first then run for 200V. The proteins will be tighter. ALSO if the % cross-linking is low then the proteins will run faster than the higher % gels