Western Blot

Goal

To extract protein samples from E. coli, run them through an SDS-PAGE gel, and use antibodies to detect specific proteins in the size-separated protein samples. This technique is useful for confirming the presence of a particular protein inside or outside of E. coli.

Protocol

Day 1

Induction and Overnight Incubation of E. coli Culture

1. Grab however many sterile erlenmeyer flasks you need for the experiment (including one for the negative control)

a. Number depends on how many E. coli variations you want to test (+1 for the negative control). For example, if you want to compare two strains of E. coli that are either uninduced (no target protein is theoretically expressed) or induced (target protein is theoretically expressed), you would need four flasks: uninduced strain 1, induced strain 1, uninduced strain 2, induced strain 2. And add one flask for the negative control: only LB broth.

b. To make sterile flasks, first grab unsterile flasks, cover the tops with aluminum foil, and autoclave them. Ideally, this should be done the day before Day 1 so that you have enough time to start and induce the cultures on Day 1, instead of waiting for the flasks to be autoclaved that same day

2. Label flasks with tape

3. Add x mL LB broth to all flasks

a. Volume used in MEE lab is typically 25-50 mL. Consult with Eric about the exact volume you would need.

4.