Labeling of Proteins in Infected Stationary Phase E. Coli Cell Cultures

This protocol is adapted from Betty Kutter. (Gel Electrophoresis Procedures, 4/1/89)

1. Check phage titer; make sure that plating bacteria/culture are dependable.
2. Grow culture of E. coli (we used W3110) in minimal media to stationary phase--the point at which the OD stops increasing.
3. Titer bacteria--diluting 100x50x50 and plating in triplicate; chill until needed.
4. Prepare labeling tubes (in a warm water bath) containing 5 uc of 35S amino acids. Tubes should be the appropriate size for the SS34 centrifuge rotor.
5. Add cells to be infected (about 3 ml) to a 50 ml sterile flask. Infect at a multiplicity of infection of 10.
6. Plate bacterial survivors and unadsorbed phage 5 or 10 minutes after infection.
7. Pulse label the cells for 4-6 minute intervals--At the beginning of interval, add 0.5 ml of infected culture to a labeling tube shaking in the warm water bath. At the end of the interval (pulse period) add 0.1 ml unlabeled 10% casamino acids.
8. 2-3 minutes after adding casamino acids, transfer labeling tubes into an ice bath.
9. Centrifuge tubes at 4000 rpm for 15 min in SS34 centrifuge rotor.
10. Pour off supernatant into radioactive sink and invert tubes in a beaker lined with Kim-wipes for 10 or 15 minutes.
11. Resuspend each pellet in 0.1 ml of SDS sample buffer containing tracking dye, 3 ul of glycerol and 1 ul of DTT.
12. Boil tubes for 3 min--make sure pellet is completely resuspended (these samples can be frozen for storage).
13. Check for radioactivity incorporated.
14. Samples are ready to load. If samples have been frozen, reboil them before loading.

(ul= microliter, uc= microCurie)

Radiation check

This protocol is adapted from Betty Kutter. (Gel Electrophoresis Procedures, 4/1/89)

1. Spot 5 ul of prepared sample onto 3mm filter disc; label discs with pencil NOT pen.
2. Place discs in a beaker of10% acetic acid (or TCA; 5 ml/disc) on ice. Swirl the beaker so the discs do not stick together.
3. After 10 minutes, transfer the discs into a beaker of 5% acetic acid (or TCA; 5 ml/disc) on ice.
4. After 10 minutes, transfer the discs into a beaker of 95% ethanol (5 ml/disc) on ice. Let them sit for 10 minutes.
5. Dry filters overnight on crinkled foil. Cover them so they do not blow away.
6. Put filters in scintillation vials and count for 2 minutes. Counts should be greater than 5000.

Preparing a 1-D Gel

1. Pick out two glass plates, one 16 cm x 20 cm, one 18.3 cm x 20 cm, and two 1.5 mm spacers that fit the plates. The plates should be free of cracks, scratches or chips.
2. Wash plates and spacers with abrasive soap and a sponge.
3. Rinse with plenty of tap water.
4. Rinse with deionized water.
5. Rinse yet again with 95% ethanol.
6. Set aside to dry.
7. Assemble plates in casting tray.
8. Add APS and TEMED to separating gel immediately before pouring the gel. APS solution must be less than one week old.
9. Pour the separating gel between the plates to a height of about 11 cm. Pour down one side of the plates to minimize bubbling. Pour DI water onto the surface of the gel. Keep the leftover separating gel in order to determine when it has polymerized. (About an hour)
10. Pour off the excess water and rinse with 95% ethanol. Let the gel dry. If gel is to be left overnight, cover gel portion with gel buffer and put in cold room; it must also be warmed up before pouring stacking gel.
11. Add APS and TEMED to stacking gel (with lower-density acrylamide) immediately before pouring the gel.
12. Pour the stacking gel to just below the top of the shortest plate. Pour down one side of the plates to minimize bubbling. Press the well comb into the stacking gel until the base of the teeth is flush with the shortest plate. Keep the leftover stacking gel in order to determine when it has polymerized. (About an hour).
13. With a sharpie, outline the wells onto the plate, using the comb as a guide.
14. Remove comb--pulling straight out to leave precise wells. Rinse the wells with deionized water.
15. Load the samples (we used 25 uL per sample), being sure to list the order of the samples. Boil the samples if they were frozen beforehand.
16. Place gel apparatus into the receptacle, hooking up both the water circulator (at 10C) and the power source (set for constant current and normal polarity--turn voltage to maximum and current to minimum.).

Running a 1-D Gel

1. Turn on the water circulator and adjust the current to 10 mA per gel until dye front reaches gradient gel. Then, turn up to 25 mA per gel until dye front is within 0.5 cm of bottom.
2. Let gel run for appropriate time, watching the dye bands so that the samples do not run off the bottom of the gel.
3. When samples are done running, remove gel from the apparatus and place in radioactive container with 10% TCA. Shake for 30 minutes.
4. After 30 minutes, pour the 10% TCA back into its original jar and pour deionized water into the gel container. Shake for 10 minutes.
5. Discard deionized water in radioactive sink.
6. Cut a piece of thick filter paper to size of gel. Thickness is important in order to avoid cracking of the gel.
7. Cover gel/paper with saran wrap and place in the protein gel dryer.
8. Open the right c.w. spigot and press the rubber mat (atop the gel) at the corners to aid in the creation of a vacuum.
9. Set timer for two hours - dryer will turn off automatically.
10. After heat goes off, break seal on dryer cover by pulling rubber hose off gel dryer and open up before turning off the water generating the vacuum. Otherwise, water will get sucked in and ruin your gels!

Recipes

10% Ammonium Persulfate
0.10g ammonium persulfate
1 ml sterile filtered DI water
Refrigerate

2D Gel Buffer
3.0 M Tris-base
0.3% SDS
adjust pH to 8.45

Acrylamide/bisacrylamide stock solution (49.5% T, 3% C)
48 g of acrylamide
1.5 g of bisacrylamide
Add DI water to a final volume of 100 ml

12% 2D separating gel
73 ml of 49.5% T, 3% C Acrylamide/bis stock
10 ml of 2D gel buffer
4 g glycerol
Add DI water to a final volume of 100 ml

Right before you pour the gel, add:
100 ul 10% APS 30 ul TEMED

Stacking gel
1 ml of 49.5% T, 3% C Acrylamide/bis stock
3.1 ml of 2D gel buffer
Add DI water to a final volume of 15 ml

Right before you pour the gel, add:
100 ul 10% APS 30 ul TEMED

Media
We supplemented basic M-9 salts with .02% casamino acids, .6% glycerol, .1% of each of the following salts: CaCl2, MgSO4, FeCl3, ZnCl2.