Protein Expression Analysis

Preparation:

1. Take enough LB/AMP plates out of fridge to warm. One plate for every 8 to 10 samples. Plates will be divided into pie shaped wedges for streaking samples. 
2. Take Amp, IPTG and 1x SDS-PAGE sample buffer out of freezer to thaw. IPTG can be placed in the fridge or on the benchtop. 
3. Label sufficient 13x100 plastic tubes to allow for the # colonies selected from each LIC plate. (Most often 2 colonies are selected for each plate. (2 colonies)x 15 plates = 30 tubes)

Procedure:

1. Add 3 ml of LB broth (medium for bacteria) containing ampicillin (final concentration of 150 ug/ml). Note: Add antibiotic each time. 
2. Using a 1 ml (1000 µl) pipet tip, select colonies (look for single colonies) by scraping part of a colony from the plate and placing the tip in the tube containing 3 ml LB/Amp broth. Remove the tips prior to placing the culture tubes in the shaking incubator. 
3. Place the cultures at 37°C in a shaking incubator (280-300rpm) for 2.5 hours until the A₆₀₀ is approximately 0.4, slightly cloudy (don't necessarily have to measure this) 
4. While the culture tubes are incubating, divide LB/AMP plates into 8 or 10 sections for streaking.
5. Remove the culture tubes from the incubator and prepare a stock culture for the positive expression clones by streaking out a loopful from each culture tube on an agar plate. 
6. Add 30 µl of 100 mM IPTG (induces bacteria to express protein) to each of the culture tubes and return the culture tubes to a shaker incubator for additional 2 hours. 
7. While waiting for induction, label 1.5 ml Eppendorf tubes. 
8. After incubation is complete, remove 400 µl from each culture tube and place in a 1.5 ml Eppendorf tbue. Centrifuge for 10 sec. at max. rpm (see note at end of procedure for processing remainder of culture) 
9. Place beaker with water on heat block to begin warming/boiling. 
10. Load gels into electrophoresis machine and take LMW out of freezer. 
11. Aspirate supernatant (next door) and resuspend the pellet in 70 µl of 1x SDS-PAGE buffer (blue color, contains B-mercaptoethanol) and vortex well. 
12. Boil for 4 mins in a beaker. Load 8 µl of sample and 5 µl of LMW to run SDS gel (4-20% precast Tris-Glycine gel(polyacrylamide) ) 
13. Stain using Invitrogen Simply Blue.