IPTG (Dioxane Free)

IPTG (isopropyl beta-D-1-thiogalactopyranoside) induces the transcription of genes from the lac and tac operons in bacteria, notably the hydrolase enzyme beta-galactosidase (ß-Gal). Once expressed, ß-Gal hydrolyzes beta-galactoside and lactose sugars into monosaccharides. IPTG is commonly used in the beta-galactosidase assay, wherein cells transfected with vector carrying the E. coli-derived lacZ gene, which encodes ß-Gal, are lysed and analyzed via the reaction substrate O-nitrophenyl-beta-D-galactopyranoside (ONPG). The ONPG substrate used in the beta-galactosidase assay turns yellow upon cleavage by ß-Gal, allowing quantitation of the transfected ß-Gal in cells and its use as a transfection control.

IPTG is also commonly used in blue/white screening experiments to determine the presence or absence of bacterial recombinant vectors following transformation. If the insert of interest is cloned into the vector's lacZ gene, it disrupts ß-Gal and prevents its expression. Bacterial colonies are grown on media containing X-gal (5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside), a substrate for ß-Gal that turns into an insoluble blue product upon being cleaved by the enzyme. Without ß-Gal, bacterial colonies exposed to X-gal cannot process the substrate and remain white, evidence that they are transformed with recombinant vector and not non-recombinant vector. These white bacterial colonies can be selected for subsequent recombinant vector amplification.

Many regulatory elements of the lac operon are used in inducible recombinant protein systems; IPTG is an effective inducer when used in the concentration range of 100 micromolar to 1.5 millimolar. Inducer concentration depends on the required strength of induction as well as the cell or plasmid genotype; for example, if the organism genotype includes lacIq, a mutation that results in the overproduction of the lac repressor, then a higher concentration of IPTG may be necessary.

Applications

• Beta-galactosidase (ß-Gal) expression
• Blue/white colony screening
• lac and tac promoter induction
• Beta-galactosidase assay