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

Before Experiment

1. Autoclave however many Erlenmeyer flasks you need for the experiment

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. Set up liquid cultures of all E. coli strains you want to use. The general contents of each culture will be: 10 mL LB broth + x uL antibiotic (or none, depends on strain) + inoculate culture with freezer stock and incubate in the 37 degrees C shaking incubator at 125 rpm overnight. Antibiotic volume depends on antibiotic stock concentration and final volume in flask.

3. During the incubation step of the induced cultures in Day 1, you will need clamps to hold the flasks down onto the incubator floor since the rotation speed will be too fast for a sticky pad to hold the flasks down. Make sure you have enough, and the correct size, clamps to perform the incubation step. Rubber bands can be used in lieu of metal rings.

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)

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. Add <10 uL E. coli overnight culture and x uL of the corresponding antibiotics (if any) into the appropriate flasks

a. Antibiotic volume depends on antibiotic stock concentration and the final volume in the flask

5. Place flasks in the 37 degrees C shaking incubator at 125 rpm until the absorbance at 600 nm reaches 0.6-0.8.

6. Check on the cultures from time to time.

a. The most effective way is to get a 13mm test tube per culture you want to measure and another tube for a negative control. Add ~3 mL culture into the tubes, and add ~3 mL LB media into the negative control tube

b. Measure on the spectrophotometer at 600 nm and record the absorbances. The numbers will allow you to estimate how long you have to wait for the cultures to be done (e.g., abs = 0.2, and E. coli takes about 30-45 min to double, so you need to wait 60-90 min (two doublings) for the culture to be ready at 0.8).

c. Pipette the 3 mL of culture back into their respective flasks, making sure not to cross-contaminate

d. Put the flasks back in the 37C shaking incubator and check again later using the same test tubes

7. Screw in the appropriate clamps into the post-induction incubator before induction in order to make sure the induced cultures are immediately placed in the incubator—putting in clamps can take a while.

8. When the cultures have reached absorbance 0.6-0.8, add x uL inducer (e.g., IPTG) to the cultures that you want to induce

a. Volume of inducer depends on the inducer stock concentration, final volume in the flask, and the desired final concentration of the inducer. Multiple inducers may be added depending on the strain. 50 uM of IPTG (final) and 1.6 mM of 100% propionate (final) are ideal for inducing E. coli strains containing bacteriocin (IPTG) and transporter (propionate) plasmids.

9. Incubate all flasks for 12-18 hours overnight in the 34 degrees C shaking incubator at 200 rpm

a. 18 degrees C shaking incubator is typically used in other labs, but we found that 34 degrees C works just as well.

Day 2

Obtaining Supernatant and Lysate Samples from Bacterial Culture

1. Add the entire volume in each overnight flask to its own appropriately-sized falcon tube (don’t include negative control). Place tubes on ice. You don’t need to transfer culture in a hood; you can just transfer normally.

2. Use “fast temp” program on swinging-bucket centrifuge (in Bio Superlab) without adding any tubes to quickly cool the centrifuge down to 4 degrees C

3. Weigh the tubes to make sure they are within 0.1 g of each other (0.5 g at worst)

a. This step ensures the centrifuge is balanced

4. Centrifuge the tubes at 3900 rpm for 40 minutes at 4 degrees C

5. Transfer supernatant into another falcon tube and label appropriately. Keep on ice.

6. Resuspend the cell pellets in x uL lysis buffer by vortexing

a. To determine the volume of lysis buffer to use, use the following ratio: 15 mL lysis buffer per 800 mL culture

b. Lysis buffer recipe (5 mL total, adjust based on how much you need)

• 150 uL 5 M NaCl (150 mM final) - chemical shelf

50 uL 100% Triton X-100 (1% final) - chemical shelf

1 mL 1 M Tris pH 8 (50 mM final) - chemical shelf

3.8 mL MilliQ water

50 uL beta-mercaptoethanol (1% final), MUST BE ADDED RIGHT BEFORE USING THE BUFFER, - flammable cabinet

50 uL protease inhibitor cocktail, MUST BE ADDED RIGHT BEFORE USING THE BUFFER, - -20C freezer