I have prepared 12% of SDS gel and a sample concentration that needs to load 24 ul in each lane to fulfill the requirement of 4uM protein concentration and the maximum volume of the lane is 20 ul. I have also prepared 2X sample loading dye. How much loading dye should I add to the sample so that it will work?
If you have 2x loading dye you need to mix the protein sample and dye in a 1:1 ratio.
As an aside, I must have run about a thousand gels or more and I’ve never before come across a “4uM requirement”. The amount of sample you load will depend on sample purity, and usually you would consider ug of protein rather than its concentration. If you have a pure protein, about 1-2ug of protein is loaded (though it depends on the size of the gel). A crude extract with multiple bands may require 10x as much, thus I do not see how a fixed 4uM requirement could work. Just measure your protein, which will typically give you a ug/ml value, and then work out what vol of sample is needed per track to give the required number of micrograms.
- Badges
- Science method