O-Propargyl-Puromycin (O-Propargylpuromycin)
(Synonyms: OP-puro) 目录号 : GC30010A clickable form of puromycin
Cas No.:1416561-90-4
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.50%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
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Animal experiment: |
Mice: 100 μL of a 20 mM solution of O-propargyl-puromycin in PBS are injected intraperitoneally into a 3-wk-old mouse, and a mouse injected with 100 μL of PBS is used as negative control. Various organs are harvested after 1 h and are fixed in formalin overnight. Organ fragments are embedded in paraffin, sectioned, and ished with xylene to remove the paraffin. After ishing with ethanol and rehydration in TBS, the tissue sections are stained with 20 μM tetramethylrhodamine(TMR)-azide. The tissue sections are counterstained with Hoechst, mounted in standard mounting media, and are then imaged by fluorescence microscopy and DIC[1]. |
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References: [1]. Liu J, et al. Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc Natl Acad Sci U S A. 2012 Jan 10;109(2):413-8. |
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O-Propargyl-puromycin (OP-puro) is a clickable form of the protein synthesis inhibitor puromycin.1 It inhibits protein synthesis in rabbit reticulocyte lysates and HEK293T cells when used at concentrations ranging from 1 to 25 µM. OP-puro is incorporated into nascent polypeptide chains where it forms covalent adducts and terminates chain elongation on the ribosome. It has been used in combination with fluorescent azides to image nascent proteins in various cells.1,2
References:
1.Liu, J., Xu, Y., Stoleru, D., et al.Imaging protein synthesis in cells and tissues with an alkyne analog of puromycinProc. Natl. Acad. Sci. USA109(2)413-418(2012)
2.Forester, C.M., Zhao, Q., Phillips, N.J., et al.Revealing nascent proteomics in signaling pathways and cell differentiationProc. Natl. Acad. Sci. USA115(10)2353-2358(2018)
| Cas No. | 1416561-90-4 | SDF | |
| 别名 | OP-puro | ||
| 化学名 | 3'-[[(2S)-2-amino-1-oxo-3-[4-(2-propyn-1-yloxy)phenyl]propyl]amino]-3'-deoxy-N,N-dimethyl-adenosine | ||
| Canonical SMILES | CN(C)C1=C2C(N([C@H]3[C@H](O)[C@H](NC([C@@H](N)CC4=CC=C(OCC#C)C=C4)=O)[C@@H](CO)O3)C=N2)=NC=N1 | ||
| 分子式 | C24H29N7O5 | 分子量 | 495.53 |
| 溶解度 | DMSO : ≥ 31 mg/mL (62.56 mM) | 储存条件 | Store at -20°C |
| 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 | 2.018 mL | 10.0902 mL | 20.1804 mL |
| 5 mM | 0.4036 mL | 2.018 mL | 4.0361 mL |
| 10 mM | 0.2018 mL | 1.009 mL | 2.018 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。
Quantification of tissue-specific protein translation in whole C. elegans using O-Propargyl-Puromycin labeling and fluorescence microscopy
Cell Rep Methods 2022 Apr 25;2(4):100203.35497499 PMC9046455
The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to quantify protein translation in C. elegans, they have been limited to using lysate from whole worms. To achieve tissue-specific quantification of ribosome activity in intact C. elegans, we report the application of O-Propargyl-Puromycin in a cuticle defective mutant followed by conjugation of an azide fluorophore for detection using fluorescent confocal microscopy. We apply this technique to quantify translation in response to heat shock, cycloheximide, or knockdown of translation factors. Furthermore, we demonstrate that O-Propargyl-Puromycin can be used to quantify translation between tissues or within a tissue like the germline. This technique is expected to have a broad range of applications in determining how protein translation is altered in different tissues in response to stress or gene knockdowns or with age.
Detection of O-Propargyl-Puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities
Biochem Biophys Res Commun 2016 May 27;474(2):247-251.27125456 10.1016/j.bbrc.2016.03.155
The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature polypeptides. The trafficking of puromycin-conjugated (puromycylated) immature polypeptides within cell has, however, remained elusive. In this study, using O-Propargyl-Puromycin (OP-Puro), the distribution of puromycylated polypeptides was assessed in HeLa cells by click chemistry. Under standard culture conditions, OP-Puro signals were detected in the cytoplasm and nucleus with the highest concentrations in the nucleolus. Intriguingly, when proteasome activities were aborted using MG132, OP-Puro signals began to accumulate at promyelocytic leukemia nuclear bodies (PML-NBs) in addition to the nucleolus. We also found promiscuous association of OP-Puro signals with SUMO-2/3 and ubiquitin at PML-NBs, but not at the nucleolus, during abortive proteasome activities. This study reveals a previously unknown distribution of OP-Puro that argues for a nuclear function in regulating immature protein homeostasis.
Treatment with surfactants enables quantification of translational activity by O-Propargyl-Puromycin labelling in yeast
BMC Microbiol 2021 Apr 20;21(1):120.33879049 PMC8056590
Background: Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-Propargyl-Puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin. Results: We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways. Conclusions: We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed.
Quantitative nascent proteome profiling by dual-pulse labelling with O-Propargyl-Puromycin and stable isotope-labelled amino acids
J Biochem 2021 Mar 5;169(2):227-236.32926143 10.1093/jb/mvaa104
Monitoring translational regulation in response to environmental signals is crucial for understanding cellular proteostasis. However, only limited approaches are currently available for quantifying acute changes in protein synthesis induced by stimuli. Recently, a clickable puromycin analogue, O-Propargyl-Puromycin (OPP), was developed and applied to label the C-termini of nascent polypeptide chains (NPCs). Following affinity purification via a click reaction, OPP allows for a proteomic analysis of NPCs. Despite its advantage, the affinity purification of NPCs using magnetic beads or resins inherently suffers from significant non-specific protein binding, which hinders accurate quantification of the nascent proteins. To address this issue, we employed dual-pulse labelling of NPCs with both OPP and stable isotope-labelled amino acids to distinguish bona fide NPCs from non-specific proteins, thereby enabling the accurate quantitative profiling of NPCs. We applied this method to dissecting translation responses upon transcriptional inhibition and quantified ∿,000 nascent proteins. We found that the translation of a subset of ribosomal proteins (e.g. RPSA, RPLP0) as well as signalling proteins (e.g. BCAR3, EFNA1, DUSP1) was significantly repressed by transcription inhibition. Together, the present method provides an accurate and broadly applicable nascent proteome profiling for many biological applications at the level of translation.
Protocol for assessing translational regulation in mammalian cell lines by OP-Puro labeling
STAR Protoc 2022 Aug 29;3(3):101654.36072758 PMC9442383
Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess translation activity in mammalian cells by incorporation of O-Propargyl-Puromycin (OP-Puro). OP-Puro is a puromycin analog that is incorporated into newly synthesized proteins and is detected by click chemistry reaction. We use OP-Puro labeling to assess translation activity between different cell types or cells under different growth conditions by confocal microscopy and flow cytometry. For complete details on the use and execution of this protocol, please refer to Hsu et al. (2021) and Hsu et al. (2022).