IPTG Induction Protocol

IPTG 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 is 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 IPTG induction protocol is generalized and will vary based on a variety of factors such as the bacterial strain, recombinant protein, and parent plasmid.

Fast IPTG induction protocol

  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.
  3. Prepare 1ml LB+AMP+1mM IPTG in a 15ml conical and prewarm to 37 C about 10min before use.
  4. 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.
  5. 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.
  6. 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.
  7. 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.).

Slow IPTG induction protocol

For slow IPTG induction protocol of protein follow fast IPTG 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.

More details from:http://genetics.wustl.edu/tslab/protocols/protein-stuff/iptg-induction/

Use in laboratory

IPTG is an effective inducer of protein expression in the concentration range of 100 μM to 1.0 mM. Concentration used depends on the strength of induction required, as well as the genotype of cells or plasmid used. If lacIq, a mutant that over-produces the lac repressor, is present, then a higher concentration of IPTG may be necessary.

In blue-white screen, IPTG is used together with X-gal. Blue-white screen allows colonies that have been transformed with the recombinant plasmid rather than a non-recombinant one to be identified in cloning experiments.

More details from: https://en.wikipedia.org/wiki/Isopropyl_%CE%B2-D-1-thiogalactopyranoside