7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin
(Synonyms: 7-二乙氨基-3-(4-马来酸亚胺苯基)-4-甲基香豆素) 目录号 : GC40882A thiol-reactive fluorescent probe
Cas No.:76877-33-3
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >95.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin is a thiol-reactive fluorescent probe. It displays excitation/emission maxima of 387/468 nm, respectively, and fluorescence intensity increases when bound to cysteine residues.
| Cas No. | 76877-33-3 | SDF | |
| 别名 | 7-二乙氨基-3-(4-马来酸亚胺苯基)-4-甲基香豆素 | ||
| 化学名 | 1-[4-[7-(diethylamino)-4-methyl-2-oxo-2H-1-benzopyran-3-yl]phenyl]-1H-pyrrole-2,5-dione | ||
| Canonical SMILES | CCN(CC)C1=CC=C(C(C)=C(C2=CC=C(N3C(C=CC3=O)=O)C=C2)C(O4)=O)C4=C1 | ||
| 分子式 | C24H22N2O4 | 分子量 | 402.4 |
| 溶解度 | Slightly soluble in DMSO, Slightly soluble in Methanol | 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 | 2.4851 mL | 12.4254 mL | 24.8509 mL |
| 5 mM | 0.497 mL | 2.4851 mL | 4.9702 mL |
| 10 mM | 0.2485 mL | 1.2425 mL | 2.4851 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。
Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies
PLoS One 2021 Feb 24;16(2):e0247689.PMID:33626080DOI:10.1371/journal.pone.0247689.
Aiming at streamlining GPCR production from E. coli inclusion bodies for structural analysis, we present a generic approach to assess and optimize refolding yield through thermostability analysis. Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding, we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). Successful expression, purification and refolding is shown for two G protein-coupled receptors (GPCR), the sphingosine-1-phosphate receptor S1P1, and the orphan receptor GPR3. Refolded receptors were subjected to lipidic cubic phase crystallization screening.
Photochemical and Structural Studies on Cyclic Peptide Models
Molecules 2018 Aug 30;23(9):2196.PMID:30200264DOI:10.3390/molecules23092196.
Ultra-violet (UV) irradiation has a significant impact on the structure and function of proteins that is supposed to be in relationship with the tryptophan-mediated photolysis of disulfide bonds. To investigate the correlation between the photoexcitation of Trp residues in polypeptides and the associated reduction of disulfide bridges, a series of small, cyclic oligopeptide models were analyzed in this work. Average distances between the aromatic side chains and the disulfide bridge were determined following molecular mechanics (MM) geometry optimizations. In this way, the possibility of cation⁻π interactions was also investigated. Molecular mechanics calculations revealed that the shortest distance between the side chain of the Trp residues and the disulfide bridge is approximately 5 Å in the cyclic pentapeptide models. Based on this, three tryptophan-containing cyclopeptide models were synthesized and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Experimental data and detailed molecular dynamics (MD) simulations were in good agreement with MM geometry calculations. Selected model peptides were subjected to photolytic degradation to study the correlation of structural features and the photolytic cleavage of disulfide bonds in solution. Formation of free sulfhydryl groups upon illumination with near UV light was monitored by fluorescence spectroscopy after chemical derivatization with 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) and mass spectrometry. Liquid cromatography-mass spectrometry (LC-MS) measurements indicated the presence of multiple photooxidation products (e.g., dimers, multimers and other oxidated products), suggesting that besides the photolysis of disulfide bonds secondary photolytic processes take place.
Cysteine residue is not essential for CPM protein thermal-stability assay
Anal Bioanal Chem 2015 May;407(13):3683-91.PMID:25772562DOI:10.1007/s00216-015-8587-4.
A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45-55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.
Design, Validation, and Application of an Enzyme-Coupled Hydrogen Sulfide Detection Assay
Biochemistry 2019 Feb 12;58(6):474-483.PMID:30547566DOI:10.1021/acs.biochem.8b01083.
Hydrogen sulfide (H2S) is a key metabolite in biosynthesis and is increasingly being recognized as an essential gasotransmitter. Owing to its diffusible and reactive nature, H2S can be difficult to quantify, particularly in situ. Although several detection schemes are available, they have drawbacks. In efforts to quantify sulfide release in the cross-linking reaction of the flagellar protein FlgE, we developed an enzyme-coupled sulfide detection assay using the Escherichia coli O-acetylserine sulfhydrylase enzyme CysM. Conversion of HS- to l-cysteine via CysM followed by derivatization with the thiol-specific fluorescent dye 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin enables for facile detection and quantification of H2S by fluorescent HPLC. The assay was validated by comparison to the well-established methylene blue sulfide detection assay and the robustness demonstrated by interference assays in the presence of common thiols such as glutathione, 2-mercaptoethanol, dithiothreitol, and l-methionine, as well as a range of anions. We then applied the assay to the aforementioned lysinoalanine cross-linking by the Treponema denticola flagellar hook protein FlgE. Overall, unlike previously reported H2S detection methods, the assay provides a biologically compatible platform to accurately and specifically measure hydrogen sulfide in situ, even when it is produced on long time scales.
Monomerization and aggregation of β-lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation
Biochim Biophys Acta Proteins Proteom 2018 Feb;1866(2):316-326.PMID:29155106DOI:10.1016/j.bbapap.2017.11.007.
β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~55Å, ~29Å and ~37Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57kcalmol-1 and 8.93kcalmol-1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~77Å and ~117Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69kcalmol-1 and 9.09kcalmol-1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.