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一、X-Gal

X-Gal也稱5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside或5-Bromo-4-chloro-3-indolyl β-D-galactoside，中文名為5-溴-4-氯-3-吲哚-β-D-半乳糖苷。分子式為C14H15BrClNO6，分子量為408.63，CAS Number 7240-90-6，純度>99%。進(jìn)口分裝。

本產(chǎn)品為白色至淺黃色粉末。

X-Gal是β-半乳糖苷酶(β-galactosidase)的顯色底物，在β-半乳糖苷酶的催化下會(huì)產(chǎn)生藍(lán)色產(chǎn)物。常用于β-半乳糖苷酶的原位染色檢測(cè)以及藍(lán)白斑篩選。

溶于DMSO，溶解度可達(dá)20mg/ml。也溶于DMF。

X-gal (also abbreviated BCIG for bromo-chloro-indolyl-galactopyranoside) is an organic compound consisting of galactose linked to a substituted indole. It is very heavily used in molecular biology.

Uses

Cloning

In gene cloning, X-gal is used to indicate whether a cell expresses the β-galactosidase enzyme, which is encoded by the lacZ gene, in a technique called blue/white screening.

X-gal is cleaved by β-galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter is then oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product. Thus, if X-gal and an inducer of β-galactosidase (usually IPTG) is contained within an agar medium on a culture plate, colonies which have a functional lacZ gene can easily be distinguished.

When the technique of cloning plasmid vector genes within bacterial cells is optimal, X-gal is used to visually locate yeast or E. coli colonies that have been transformed by the desired plasmid vector in a blue-white screen. E. coli bacteria, which cannot produce the enzyme β-galactosidase (coded by lacZ gene of the lac operon), are transformed by absorbing the plasmid vectors, which contain an insert, in the lacZ open reading frame. After the transformation process, the bacteria is spread on nutrient agar plates, which mostly contain antibiotics as well. Most commercially available vectors contain an antibiotic-resistant gene. Successfully transformed bacteria has a truncated β-galactosidase gene, causing white colonies on the plate. Bacteria transformed by empty vectors, which do not contain an insert in the lacZ open reading frame, are now able to produce the enzyme β-galactosidase which can then cleave the X-gal present within the nutrient agar, resulting in a blue colony. Bacteria colonies that grow from bacteria that were not transformed do not contain this antibiotic-resistance, and thus, die. The plasmid vectors can also be coded to disrupt a different bacteria's ability to produce β-galactosidase, causing the desired bacteria colonies to grow to be white and non-transformed colonies to grow to be blue. This is the case with many commercially available cloning vectors, such as Promega's pGem-T Vectors, which carry lacZα, a truncated form of β-galactosidase, and require specific E. coli hosts a strain (such as DH5α) to achieve α-complementation.

Reporter

The lacZ gene may be used as a reporter in combination with growth media containing X-gal. In two-hybrid analysis for example, it is necessary to distinguish between those yeast or bacteria in which there is a successful interaction, leading to the binding of an activation domain to a promoter, and those in which there is not. If the promoter is linked to a lacZ gene, the production of β-galactosidase will be indicated by the production of blue pigment by colonies that host a successful interaction.[1] Due to its manual nature, this technique is limited to situations in which the number of colonies that must be distinguished is less than around 106.[1] The successful cleavage of X-gal also creates a noticeably foul odor due to the volatilization of indole.

Water testing

In addition to use in molecular biology, X-Gal is used to determine E. coli and coliform content in drinking water samples.

二、IPTG

中文品名: 異丙基-β-D-硫代吡喃半乳糖苷

商品名稱: Isopropyl β-D-1-Thiogalactopyranoside

英文簡(jiǎn)稱: IPTG

產(chǎn)品別名: Isopropyl β-D-Thiogalactoside

通用CAS : 367-93-1

產(chǎn)品純度: Sigma ≥99%, Merck ≥98% ，INALCO≥99%

產(chǎn)品性狀: 白色或白色粉末,溶于水后呈清亮無色液體

分 子式: C9H18O5S

分 子量: 238.30

保存溫度: 2-8°C冷藏保存 ,配制好后保存于-20°C，室溫可放置一個(gè)月

主要作用：異丙基硫代半乳糖苷(IPTG)是一種作用極強(qiáng)的誘導(dǎo)劑,不被細(xì)菌代謝而十分穩(wěn)定,因此被實(shí)驗(yàn)室廣泛應(yīng)用

IPTG是β–半乳糖苷酶的活性誘導(dǎo)物質(zhì)�；�于這個(gè)特性，當(dāng)pUC系列的載體DNA（或其他帶有l(wèi)acZ 基因載體DNA）以lacZ 缺失細(xì)胞為宿主進(jìn)行轉(zhuǎn)化時(shí)、或用M13噬菌體的載體DNA進(jìn)行轉(zhuǎn)染時(shí)，如果在平板培養(yǎng)基中加入X–Gal和IPTG，由于β–半乳糖苷酶的α–互補(bǔ)性，可以根據(jù)是否呈現(xiàn)白色菌落（或噬菌斑）而方便地挑選出基因重組體。此外，它還可以作為具有l(wèi)ac或tac等啟動(dòng)子的表達(dá)載體的表達(dá)誘導(dǎo)物使用。

Isopropyl β-D-1-thiogalactopyranoside, abbreviated IPTG, is a molecular biology reagent.

This compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon. Unlike allolactose, the sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from "eating up" or degrading the inductant; therefore the IPTG concentration remains constant. For induction, a sterile 1 M solution of IPTG is typically added by 1:1000 dilution into a logarithmically growing bacterial culture. Higher concentrations can be used.

IPTG induces the transcription of the gene coding for beta-galactosidase, a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. One advantage of IPTG for in vivo studies is that since it cannot be metabolized by E. coli its concentration remains constant and the rate of expression of lac p/o-controlled genes, is not a variable in the experiment. IPTG intake is independent on the action of lactose permease, since other transport pathways are also involved.

In cloning experiments, colonies that have been transformed with the recombinant plasmid rather than a non-recombinant need to be identified. X-gal is a substance that can be metabolised by beta-galactosidase to produce a blue product. Thus cells expressing beta-galactosidase grown in the presence of X-gal and IPTG (to induce the expression) will turn blue. Where a DNA fragment has been inserted into the LacZ (one of the genes for beta-galactosidase) there will be no action upon X-gal and the cells will not turn blue, thus identifying the cells that carry recombinant plasmid rather than non-recombinant plasmid.

Many regulatory elements of the lac operon are used in inducible recombinant protein systems; IPTG is an effective inducer in the concentration range of 100 μM to 1.5 mM.

三、X-α-Gal

CAS:107021-38-5

英明；5-Bromo-4-chloro-3-indoxyl-α-D-galactopyranoside;X-α-D-Galactoside

分子式：C14H15BrCNO6

分子量：408.64 g/mol

報(bào)告基因Mel表達(dá)后，在含X-α-Gal的平板上顯藍(lán)色

溶解性：清澈溶液（2%in DMF）

儲(chǔ)存：4℃ 避光避潮；長期存放-20℃

用途：X-α-半乳糖苷酶的作用底物，用于篩選含X-α-半乳糖苷酶基因的陽性酵母或細(xì)菌菌株。在組織化學(xué)中用于酶活性的檢測(cè)。

貯液：

A)涂布于預(yù)制平板：溶解24mg X-α-gal 于6 mL DMF中得到終濃度為4 mg/mL的溶液。

B)直接加入瓊脂中：溶解60mg X-α-gal 于3 mL DMF中得到終濃度為20 mg/mL的溶液。

參考用法：

A)涂布于預(yù)制平板

1.涂布200ul(15cm) 或者100ul（10cm） X-α-gal 貯液于預(yù)制平板上。

2. 放于37 oC培養(yǎng)箱至液體被吸收（約4小時(shí)）。

3. 將轉(zhuǎn)化細(xì)菌或酵母涂于平板上并于合適溫度培養(yǎng)至藍(lán)色菌落出現(xiàn)。

B)直接加入瓊脂中

1. 將已滅菌瓊脂培養(yǎng)基冷卻至55-50 oC。

2. 在已冷卻的培養(yǎng)基中加入20 mg/ ml X-α-Gal貯液，混勻，快速倒平板。

四、X-gluc

CAS：18656-96-7

分子式：C14H13BrClNO7.C6H13N

分子量：521.8

外觀：白色粉末

溶解性：清澈溶液（2％in DMF）

用途：

檢測(cè)大腸桿菌中uidA基因（β-葡萄糖苷酶基因β-glucuronidase, GUS）的底物。95%的大腸桿菌具有β-葡萄糖苷酶基因，用這種發(fā)色底物的培養(yǎng)基可以檢測(cè)以及定量食品樣本如肉、奶制品以及貝類中的大腸桿菌數(shù)量，以及在臨床上檢測(cè)尿路感染。國際上普遍采用該產(chǎn)品取代傳統(tǒng)方法以精確檢測(cè)飲用水中的大腸桿菌數(shù)量，該方法大大降低了假陽性和假陰性。該產(chǎn)品也用于快速檢測(cè)植物中GUS基因融合標(biāo)記。

β-葡糖醛酸酶（β-glucuronidase (GUS)）基因檢測(cè)的顯色底物。