IPTG Induction and Extraction of Proteins from Bacteria
Prepared by Swathi Arur and Sudhir Nayak
Induction in bacteria can be performed using one of two basic methods. Fast induction does not work for all proteins and can give you suboptimal yields. Slow induction can enhance the solubility of some proteins. The method that’s best for you will depend on your particular protein and the application. If you want optimal solubility both should be tested before scaling up. This protocol is generalized and will vary based on a variety of factors such as the bacterial strain, recombinant protein, and parent plasmid.
1) From a relatively fresh plate (<4 weeks) pick a colony and grow O/N at 30C (or 37C) in 1-2ml LB+AMP (or other selection) in a 15ml snap cap tube on a rotator or shaker.
2) Dilute 1:50 (1:100 if 37C O/N) in 2ml LB+AMP and grow 3-4 hours at 37 C in 15ml snap cap tube in a rotator.
4) Prepare 1ml LB+AMP+1mM IPTG in a 15ml conical and prewarm to 37 C about 10min before use.
5) After 3-4hrs remove 1ml from tubes at 37deg C and place in labeled 1.5ml tubes. Spin at max, 30sec, RT, and remove supe. Freeze pellet at -20 until needed. THIS IS THE UNINDUCED CONTROL.
6) Add prewarmed 1ml LB+AMP+1mM IPTG to 15ml snap cap tube and return to 37 C for 3-4 hours. This will get the final volume back to 2ml and the final concentration of IPTG to 0.5mM.
7) After 3-4hrs transfer 1ml from induced sample to labeled 1.5ml tubes and spin at max, 30sec, RT, and remove supe. Freeze pellet at -20 until needed. THIS IS THE INDUCED SAMPLE.
8) Sample preparation for SDS-PAGE: Add 100ul of 1X loading buffer (see solutions below) with 1% BME to uninduced and induced samples. Vortex for 10sec to 1min or until there are no clumps of bacteria. Boil 3-5min, spin at max, 30sec, RT, and load 5-25ul (usually 10ul) depending on gel (amount of protein, size of pellet, Western, etc.).
For slow induction of protein follow fast induction protocol with the following changes:
6) Add 20 C 1ml LB+AMP+1mM IPTG to 15ml snap cap tube and incubate rotating or shaking at 20 C for 12-16 hours. This will get the final volume back to 2ml and the final concentration of IPTG to 0.5mM.
7) After 12-16hrs transfer 1ml from induced sample to labeled 1.5ml tubes and spin at max, 30sec, RT, and remove supe. Freeze pellet at -20 until needed. THIS IS THE INDUCED SAMPLE.
NOTES for induction:
*Induction times vary from 2-5hrs.
*IPTG can be varied from 0.1-1.0M.
*If you boil your sample too long they will become viscous from total release of cellular DNA. You can still use them if you can find an area of low viscosity, however, its usually better just to repeat the experiment.
Extraction of Soluble Proteins
1) Wash the bacterial pellet with 2mls of ice cold STE (10mM Tris, pH 8.0; 150mM Nacl; 1mM EDTA) once.
2) Resuspend the bacterial pellet (from a 10ml induced culture) in 800ul of STE containing 100ug/ml of Lysozyme (added immediately prior to resuspension)
3) Incubate on ice for 15 minutes.
4) Add DTT to a final concentration of 5mM ( we have stocks at 1M in –20).
5) Add protease inhibitors.
6) Bacteria are then lysed by the addition of N-laurylsarcosine (Sarkosyl) from a 10% (w/v) stock in STE. The final concentration of N-lauryl sarcosine should be 1.5%.
7) Sonicate the cells in the small bath sonicator with for 2 cycles (6min each). The sonicator automatically turns off after 6 minutes.
8) Centrifuge the lysate for 5min at 4 C.
9) Transfer the supernatant to a new eppendorf tube and add Triton X-100 (from a 10% stock made in STE) to a final concentration of 2%.
10) Take 100ul of Ni-NTA agarose gel and wash twice by centrifugation with ice-cold PBS at 1000rpm for 1min. each
11) Add the beads to the eppendorf tube containing the lysate and the Triton X-100 tube and incubate on a nutator or rocker at RT for 30-60min.
12) Wash the beads 4X with 1ml ice-cold PBS containing 20mM imidiazole at 1000rpm for 1minute each.
13) Add 1X loading buffer with 1% BME, boil 3min, and analyze on SDS-PAGE.
NOTES for extraction:
*This basic protocol will work for FLAG, GST, and 6HIS epitope tags. It has not been tested for MBP, which does not respond will to detergents.
*The inclusion of Imidiazole is specific to 6HIS tags.
*TWEEN20 at 0.1-1% can also be incorporated into the wash buffer to reduce background if required.
Loading Buffer (To make a 4X Stock)
50 mM Tris-HCl; pH 6.8 (2.0 ml 1M Tris-HCl; pH 6.8)
2% SDS (0.8 g SDS)
10% Glycerol (4.0 ml 10% Glycerol)
12.5 mM EDTA (1.0 ml 0.5 M EDTA)
0.02 % Bromophenol Blue (8 mg Bromophenol Blue)
Add fresh 2(or beta)-mercaptoethanol (BME) to 1% before using.