IPTG (Isopropyl β-D-thiogalactoside) (Synonyms: Isopropyl βthiogalactoside)

This plan only provides a guide, please modify it to meet your specific needs.

1. Solution preparation

(1) Prepare storage solution: Equilibrate the cryopreserved solid to room temperature, use sterile water to dissolve IPTG, prepare a storage solution with a concentration of 1 M, and filter and sterilize.

Note: Unused storage solution should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freezing and thawing.

(2) Working solution: The usually used IPTG concentration should be between 0.1 and 1 mM.

Note: Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration yourself to explore. The working fluid must be prepared and used immediately.

2. Use IPTG to induce the expression of recombinant PcOSM in E.coli ^[1] (from the literature, for reference only)

(1) Transformation: Use pET 100/D-TOPO vector containing recombinant PcOSM to transform BL-21 competent cells.

(2) Inoculation and culture: Add ampicillin (final concentration 100 µg/mL) to 5 mL LB liquid medium, use a sterile pipette tip to inoculate BL-21 E.coli cells carrying recombinant PcOSM, and inoculate at 37°C Incubate with vigorous shaking at 220 rpm until OD₆₀₀ reaches 0.6 to 0.8.

(3) IPTG induction: Add IPTG (final concentration 1 mM) to the culture for induction.

(4) Harvest cells: Continue to culture at the post-induction temperature conditions for 8 hours or overnight to maximize the production of target proteins. Harvest cells by centrifugation at 4,000g for 20 minutes at 4°C, remove supernatant and retain cell pellet.

Note: This experimental protocol only provides a reference for the use of IPTG to induce protein expression experiments. The optimal IPTG concentration and induction time may need to be optimized for specific proteins. Please perform necessary control experiments to evaluate induction efficiency and cytotoxicity. Always ensure to use sterile technique.

References:

[1] Geetha R G, Krishnankutty Nair Chandrika S, Saraswathy G G, et al. ROS Dependent Antifungal and Anticancer Modulations of Piper colubrinum Osmotin[J]. Molecules, 2021, 26(8): 2239.