IPTG誘導大腸桿菌蛋白表達實驗流程

誘導實驗
 
1. Inoculate 10 ml of LB containing the appropriate antibiotic (and 1% glucose,if desired) with 500 μl of the overnight culture.
 
2. Grow two hours at 37°C with shaking. OD600 should be about 0.5-0.8 (midlog).
 
3. Split the culture into two 5 ml cultures. Add IPTG to a final concentration of 0.5-1 mM to one of the cultures. You will now have two cultures: one induced, one uninduced.
 
4. Remove a 500 μl aliquot from each culture, centrifuge at maximum speed in a microcentrifuge for 30 seconds, and aspirate the supernatant.
 
5. Freeze the cell pellets at -20°C. These are the zero time point samples.
 
6. Continue to incubate the cultures at 37°C with shaking. Take time points for each culture every hour for 4 to 6 hours.
 
7. For each time point, remove 500 μl from the induced and uninduced cultures and process as described in Steps 4 and 5.
 

分析誘導實驗結果
 
Have the following materials on hand before starting:
• Lysis Buffer
• 1X and 2X SDS-PAGE sample buffer
• Reagents and apparatus to perform SDS-PAGE electrophoresis
• Boiling water bath

 
樣品制備

Once you have finished your pilot expression, you are ready to analyze the samples you have collected. Before starting, prepare SDS-PAGE gels.
 

Note: If you wish to analyze your samples for soluble protein.

 
1. Thaw the samples (from Pilot Expression, Steps 5 and 7, previous page) and resuspend each cell pellet in 80 μl of 1X SDS-PAGE sample buffer.
 
2. Boil 5 minutes and centrifuge briefly.
 
3. Load 5-10 μl of each sample on an SDS-PAGE gel and electrophorese. Save your samples by storing them at -20°C.
 

判斷蛋白可溶性
 
1. Thaw and resuspend each cell pellet in 500 μl of Lysis Buffer.

2. Freeze sample in dry ice or liquid nitrogen and then thaw at 42°C. Repeat 2 to 3 times.
 

Note: To facilitate lysis, you may need to add lysozyme or sonicate the cells.

 
3. Centrifuge samples at maximum speed in a microcentrifuge for 1 minute at +4°C to pellet insoluble proteins. Transfer supernatant to a fresh tube and store on ice.
 
4. Mix together equivalent amounts of supernatant and 2X SDS-PAGE sample buffer and boil for 5 minutes.
 
5. Add 500 μl of 1X SDS-PAGE sample buffer to the pellets from Step 3 and boil 5 minutes.
 
6. Load 10 μl of the supernatant sample and 5 μl of the pellet sample onto an SDS-PAGE gel and electrophorese.
 

結果分析
 
To determine the success of your expression experiment, you may want to perform the following types of analyses:
 
1. Stain the polyacrylamide gel with Coomassie blue and look for a band of increasing intensity in the expected size range for the recombinant protein.Use the uninduced culture as a negative control.
 
2. Perform a western blot to confirm
 
 
蛋白表達
 
We generally scale-up expression to a 50 ml bacterial culture for purification using a 2 ml Ni-NTA column. Depending on the expression level of your recombinant fusion protein, you may need to adjust the culture volume to bind the maximum amount of recombinant fusion protein to your column.
 
To grow and induce a 50 ml bacterial culture:

1. Inoculate 10 ml of S.O.B. or LB containing the appropriate antibiotic with 500 μl of the culture of the transformation reaction.
 
2. Grow overnight at 37°C with shaking (225-250 rpm) to OD600 = 1-2.
 
3. The next day, inoculate 50 ml of S.O.B. or LB containing the appropriate antibiotic with 1 ml of the overnight culture.
 

Note: You can scale up further and inoculate all of the 10 ml overnight culture into 500 ml of medium, but you will need to adjust the bed volume of your Ni-NTA column accordingly.

 
4. Grow the culture at 37°C with shaking (225-250 rpm) to an OD600 = ~0.5 (2-3 hours). The cells should be in mid-log phase.
 
5. Add 0.5-1 mM IPTG to induce expression.
 
6. Grow at 37°C with shaking until the optimal time point determined by the pilot expression is reached. Harvest the cells by centrifugation (3000 x g for 10 minutes at +4°C).
 
 7. Proceed to purification or store the cells at -80°C for future use.