Gently mix the Unstained Protein Ladder, Broad Range (NEB #P7703 ) by gently vortexing or pipetting up and down several times.
- Incubate at 95°C-100ºC for 5 minutes.
- Centrifuge quickly.
- Aliquot in to single use aliquots and store at -20C. Do not boil the aliquots again.
- Load 5 μl of P7703 in a gel lane.
- To ensure uniform mobility, load an equal volume of 3X Blue SDS Loading Buffer (NEB #B7703) into any unused wells.
- Run gel using constant voltage, according to manufacturer’s specifications.
- Run until dye front reaches bottom of gel, but does not run off.
- Stain and de-stain gel with protein stain of choice, following manufacturer’s instructions (i.e. Coomassie Brilliant Blue, Simply BlueTM Safe Stain, or GelCodeTM Blue Safe Stain)
Note: Do not boil the aliquots a second time. The recommended load for (NEB #P7703) is 5 μl. Loading less than this amount may result in less intense bands.