Everyone knows that IPTG [CAS: 367-93-1] has a wide range of uses in biological laboratories. So, in the field of biochemical research, what are the uses of IPTG that everyone knows about? Let me introduce it to you below!

1. IPTG is mainly used as an inducer to induce protein expression and lactose operon expression.

IPTG is usually used in combination with X-GAL [CAS: 7240-90-6] for screening blue and white colonies.

3. IPTG is also used in fermentation extraction and cell culture.

4. IPTG can also be used in vaccine development, E. coli expression of target proteins, and cell recombination expression research.

IPTG induction principle

first

E. The lactose operon (element) of coli contains three structural genes: Z, Y, and A, encoding galactosidase, osmotic enzyme, and acetyltransferase, respectively. In addition, there is a manipulation sequence O, a promoter sequence P, and a regulatory gene I. The I gene encodes a repressor protein, which binds to the O sequence, causing the operon (element) to be suppressed and in a closed state. There is also a metabolite gene activator protein (CAP) binding site upstream of the initiation sequence P. The regulatory region of lac operon is composed of P sequence, O sequence, and CAP binding site. The coding genes of the three enzymes are regulated by the same regulatory region, achieving coordinated expression of gene products.

secondly

In the absence of lactose, the lac operon (element) is in a repressed state. At this point, the Lac repressor protein expressed under the manipulation of the PI promoter sequence binds to the O sequence, hindering the binding of RNA polymerase to the P sequence and inhibiting transcription initiation. When lactose is present, the lac operon (element) can be induced. In this operon (meta) system, the true inducer is not lactose itself. Lactose enters cells through β- Galactosidase catalyzes the conversion to lactose. The latter acts as an inducer molecule to bind to a repressor protein, causing a conformational change in the protein, resulting in the dissociation of the repressor protein from the O sequence and transcription. The action of isopropylthiogalactoside (IPTG) is the same as that of lactose, and it is a highly potent inducer that is not metabolized and is very stable. Therefore, it is widely used in laboratories.

Usage

Firstly, prepare IPTG into a 23.83 mg/ml (100 mM) aqueous solution, filter and sterilize it, and store it. Then add 100 ml of agar medium μ The above solution of l, 200 μ L of X-Gal (20 mg/ml of dimethylformamide (DMF) solution) and 100 μ Amp (100 mg/ml) was prepared into IPTG, X-Gal, and Amp agar plates. When DNA fragments are inserted into * pUC series vectors (or other vectors containing lacZ and Amp genes), and then transformed into * lacZ deficient cells, the aforementioned IPTG, X-Gal, and Amp agar plates can be coated to conveniently select gene recombinants (white represents gene recombinants with DNA insertion fragments) based on the blue-white color of the growing cells.

effect

Inducing the expression of exogenous genes is commonly used in prokaryotic expression systems to increase their expression levels, stabilize their products, and have the advantages of easy identification and purification.

As a large domestic manufacturer of glycoside series products, Huibai Reagent supplies high-purity IPTG with a purity of 99% to 101% to a large number of scientific research institutions