X-Gal
(Synonyms: 5-溴-4-氯-3-吲哚半乳糖苷,BCIG) 目录号 : GC15620
X-Gal被β-半乳糖苷酶水解成蓝色，因此常用于转基因筛选试验，如β-半乳糖苷酶的蓝白斑筛选或原位染色试验。
Cas No.:7240-90-6
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
X-gal储备溶液配置：
用二甲基甲酰胺溶解X-gal制备20mg/ml存储溶液。称取20g X-gal，加入1L二甲基甲酰胺(DMF)，溶解后装入瓶中，用铝箔包好避光，-20℃保存。不需过滤除菌。
成年小鼠脑切片X-gal染色[1]：
1. 小鼠用PBS灌注5分钟，然后用4% PFA/PBS灌注5分钟。
2. 将小鼠脑解剖出来，用4% PFA/PBS在4℃下固定4小时。
3. 固定脑用PBS洗涤三次，然后用20%的蔗糖孵育过夜(或直到样品下沉)，然后用30%的蔗糖孵育过夜。
4. 大脑安装在低温模具上，并在-80℃保存。
5. 使用低温恒温器制作40 μm的cyro切片，并安装在玻片上。
6. 玻片用染色缓冲液在室温下洗涤10分钟。
7. 玻片加入1 mg/ml X-gal，在37℃下加入5 mM铁氰化钾和5 mM铁氰化钾的染色缓冲液中孵育，直至显色。这一步通常需要3个小时才能完成。不要让样品干燥。每张玻片使用100-200 μl X-gal溶液。
8. 用PBS洗涤染色样品三次(每次5分钟)。
9. 载玻片用系列乙醇(50%，75%，90%，100%，各2分钟)脱水。
10. 载玻片用中性树胶封片剂保存，可以在室温下储存或者用于成像。
Reference:
[1]. Kokubu H, Lim J. X-gal Staining on Adult Mouse Brain Sections. Bio Protoc. 2014;4(5):e1064. doi: 10.21769/bioprotoc.1064. Epub 2014 Mar 5. PMID: 27390760; PMCID: PMC4932900.
X-Gal is hydrolyzed blue by β-galactosidase, so it is often used in transgenic screening tests, such as blue-white spot screening or in situ staining assay for β-galactosidase [1-2].
X-gal (1 mg/ml / 0.3 mg/ml) staining is also used to analyze protein expression pattern. It can be used to detect expression pattern of beta-galactosidase in vivo [3-4]. X-gal (1 mg/ml) can also be used to visualize LacZ activity in mouse embryos and adult mice[5-6].
References:
[1]. Green MR, Sambrook J. Screening Bacterial Colonies Using X-Gal and IPTG: α-Complementation. Cold Spring Harb Protoc. 2019 Dec 2;2019(12). doi: 10.1101/pdb.prot101329. PMID: 31792144.
[2]. Trimborn L, Hoecker U,et,al. A Simple Quantitative Assay for Measuring β-Galactosidase Activity Using X-Gal in Yeast-Based Interaction Analyses. Curr Protoc. 2022 May;2(5):e421. doi: 10.1002/cpz1.421. PMID: 35567769.
[3]. Kokubu H, Lim J. X-gal Staining on Adult Mouse Brain Sections. Bio Protoc. 2014;4(5):e1064. doi: 10.21769/bioprotoc.1064. Epub 2014 Mar 5. PMID: 27390760; PMCID: PMC4932900.
[4]. Burn SF. Detection of β-galactosidase activity: X-gal staining. Methods Mol Biol. 2012;886:241-50. doi: 10.1007/978-1-61779-851-1_21. PMID: 22639266.
[5]. Watanabe-Takano H, Fukumoto M, et,al. Protocol for whole-mount X-gal staining combined with tissue clearing in embryo and adult mouse using CUBIC. STAR Protoc. 2022 Jan 20;3(1):101127. doi: 10.1016/j.xpro.2022.101127. PMID: 35118431; PMCID: PMC8792446.
[6]. Gierut JJ, Jacks TE, et,al. Whole-mount X-Gal staining of mouse tissues. Cold Spring Harb Protoc. 2014 Apr 1;2014(4):417-9. doi: 10.1101/pdb.prot073452. PMID: 24692489; PMCID: PMC4169236.
X-Gal被β-半乳糖苷酶水解成蓝色，因此常用于转基因筛选试验，如β-半乳糖苷酶的蓝白斑筛选或原位染色试验[1-2]。
X-gal (1mg /ml / 0.3 mg/ml)染色法可以用于分析蛋白表达谱。它可用于检测体内β -半乳糖苷酶的表达模式[3-4]。X-gal (1 mg/ml)也可用于观察小鼠胚胎和成年小鼠的LacZ活性[5-6]。
| Cas No. | 7240-90-6 | SDF | |
| 别名 | 5-溴-4-氯-3-吲哚半乳糖苷,BCIG | ||
| 化学名 | (2S,3R,4S,5R,6R)-2-[(5-bromo-4-chloro-1H-indol-3-yl)oxy]-6-(hydroxymethyl)oxane-3,4,5-triol | ||
| Canonical SMILES | C1=CC(=C(C2=C1NC=C2OC3C(C(C(C(O3)CO)O)O)O)Cl)Br | ||
| 分子式 | C14H15BrCINO6 | 分子量 | 408.63 |
| 溶解度 | ≥ 109.4 mg/mL in DMSO, ≥ 3.7 mg/mL in EtOH with ultrasonic and warming | 储存条件 | store at -20°C,protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.4472 mL | 12.236 mL | 24.472 mL |
| 5 mM | 0.4894 mL | 2.4472 mL | 4.8944 mL |
| 10 mM | 0.2447 mL | 1.2236 mL | 2.4472 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Screening Bacterial Colonies Using X-Gal and IPTG: 伪-Complementation
Cold Spring Harb Protoc 2019 Dec 2;2019(12).31792144 10.1101/pdb.prot101329
Many plasmid vectors (e.g., the pUC series, Bluescript, pGem, and their derivatives) carry a short segment of Escherichia coli DNA containing the regulatory sequences and the coding information for the first 146 amino acids of 尾-galactosidase. Vectors of this type are used in host cells that express the carboxy-terminal portion of 尾-galactosidase. Although neither the host-encoded fragments nor the plasmid-encoded fragments of 尾-galactosidase are themselves active, they can associate to form an enzymatically active protein. This type of complementation, in which deletion mutants of the operator-proximal segment of the lacZ gene are complemented by 尾-galactosidase-negative mutants that have the operator-proximal region intact, is called 伪-complementation. The lac+ bacteria that result from 伪-complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate X-Gal. However, insertion of a fragment of foreign DNA into the polycloning site of the plasmid almost invariably results in production of an amino-terminal fragment that is no longer capable of 伪-complementation. Bacteria carrying recombinant plasmids therefore form white colonies. To screen bacterial colonies, the chromogenic substrate X-Gal and the gratuitous inducer IPTG are mixed with suitable dilution of a culture, combined with molten top agar, and then spread on agar plates containing the appropriate antibiotic. The efficiency of transformation is slightly higher when the bacteria are plated in top agar rather than on the surface of agar plates. Perhaps the transformed bacteria prefer the slightly anaerobic state within the soft agar or the isosmolarity provided by the agar medium.
Detection of 尾-galactosidase activity: X-Gal staining
Methods Mol Biol 2012;886:241-50.22639266 10.1007/978-1-61779-851-1_21
X-Gal staining is a rapid and convenient histochemical technique used to detect reporter gene expression. A prerequisite is the creation or acquisition of transgenic reporter mouse lines, in which the bacterial LacZ gene has been knocked into the gene of interest or placed under the control of regulatory elements corresponding to the gene of interest. Expression is marked by a dark blue stain and can be detected at the single cell level, providing a robust visual readout of gene expression in the developing kidney. Here, we describe the methodology, applications, and limitations of this technique.
Methods to detect biomarkers of cellular senescence: the senescence-associated beta-galactosidase assay
Methods Mol Biol 2007;371:21-31.17634571 10.1007/978-1-59745-361-5_3
Most normal human cells undergo cellular senescence after accruing a fixed number of cell divisions, or are challenged by a variety of potentially oncogenic stimuli, in culture and most likely in vivo. Cellular senescence is characterized by an irreversible growth arrest and certain altered functions. Senescent cells in culture are identified by their inability to undergo DNA synthesis, a property also shared by quiescent cells. Several years ago, we described a biomarker associated with the senescent phenotype, a senescence associated beta-galactosidase (SA-beta-gal), which is detected by histochemical staining of cells using the artificial substrate X-Gal. The presence of the SA-beta-gal biomarker is independent of DNA synthesis and generally distinguishes senescent cells from quiescent cells. The method to detect SA-beta-gal is a convenient, single cell-based assay, which can identify senescent cells even in heterogeneous cell populations and aging tissues, such as skin biopsies from older individuals. Because it is easy to detect, SA-beta-gal is currently a widely used biomarker of senescence. Here we describe a method to detect SA-beta-gal in detail, including some recent modifications.
The X-Gal caution in neural transplantation studies
Cell Transplant 2000 Sep-Oct;9(5):657-67.11144962 10.1177/096368970000900510
Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g.. pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-Gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-Gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli beta-gal activity.
Protocol for whole-mount X-Gal staining combined with tissue clearing in embryo and adult mouse using CUBIC
STAR Protoc 2022 Jan 20;3(1):101127.35118431 PMC8792446
Here we describe an optimized protocol for X-Gal staining of tissue clearing embryo and adult mouse using CUBIC. The activity of LacZ knock-in reflecting endogenous expression of genes of interest in the whole body can be visualized by X-Gal staining. This protocol is suitable for examining the developmental stage-specific expression of genes of interest spatially and temporally. For complete details on the use and execution of this protocol, please refer to Watanabe-Takano et al. (2021).