NEBExpress Cell-free E.coli Protein Synthesis SDS-PAGE Protocol
- Combine 2 µL of a NEBExpress™ Cell-free E. coli Protein Synthesis System reaction with 6 µL of SDS-PAGE Blue Loading Buffer (NEB #B7703), and 10 µL H2O. Also prepare a negative control sample.
- Incubate at 100°C for 3-5 minutes.
- Load 3 µL of the Unstained Protein Standard (NEB #P7717) into the first lane.
- After a quick microcentrifuge spin, load samples directly on to the gel. To ensure uniform mobility, load an equal volume of SDS-PAGE Blue Loading Buffer into any unused wells.
- Run the gel according to the manufacturer’s recommendations.
- Stain with Coomassie Blue or another stain as directed or proceed to Western Blot.
After staining, the target protein is typically observed as a unique band, absent in the negative control reaction. However; sometimes, the target has the same apparent molecular weight as an endogenous protein. In this case, the target protein will enhance or “darken” the co-migrating band.