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Quality Control & SDS

View current batch:

Purity: >98.50%

实验参考方法

WSP-1 Usage Instructions:

Preparation of Stock Solution: Remove the product from the refrigerator and allow it to equilibrate at room temperature. Centrifuge at low speed for 3-5 minutes. Add an appropriate amount of anhydrous DMSO (Dimethyl Sulfoxide) to the vial and dissolve thoroughly to prepare a 5mM stock solution. Divide the solution into single-use aliquots and store them in the freezer, avoiding repeated freeze-thaw cycles. The stock solution should remain stable for at least three months.

For specific working concentrations, please refer to the literature or adjust as needed based on your experimental requirements.

Protocol forfor Fluorescent Detection of Intracellular H₂S (Fluorescence Microscopy Examination) by WSP-1^[1]:

1.Culture HASMCs until they reach approximately 90% confluence. 24 hours before the experiment, seed the cells on 8-well culture slides at a density of 30,000 cells per well and incubate for 24 hours.
2.After 24 hours, allow the cells to adhere, replace the culture medium with 400 μL of WSP-1（100 μM）working solution, and incubate at 37℃ for 30 minutes.
3.Replace the WSP-1 working solution with 380 μL of standard buffer and add 20 μL of the required concentration of H₂S donor compound.
4.When WSP-1 reacts with hydrogen sulfide, a detectable fluorescence signal is released at Ex/Em = 465/515 nm. After incubating for 1 hour, wash the cells with 400 μL of DPBS.
5.Remove the DPBS, and at room temperature, add 200 μL of Bouin's solution to each well to fix the cells for 10 minutes. Remove excess Bouin's solution and wash the cells twice with 400 μL of DPBS.
6.Use a fluorescence microscope to examine and capture the fluorescence signals.

This protocol only provides a guideline, and should be modified according to your specific needs.

Protocol for Visualizing Intracellular H₂S Using WSP-1^[2]:

1.The roots of seedlings after treatments were transferred to 20 mM Hepes-NaOH (pH 7.5) buffer solution containing 15 μM of WSP-1.
2.After being incubated in darkness at 25℃ for 40 min, the roots were washed with distilled water three times and were visualized immediately by a fluorescence microscope with a 465/515 nm and an excitation/emission filter set.
3.For the detection of WSP-1 fluorescence in different reactive sulfur species, WSP-1 with final concentration of 15 μM were added into the following solutions, SDS (sodium dodecyl sulfate, 2 mM), NaHSO₄ (2 mM), NaHSO₃ (2 mM), Na₂SO₄ (2 mM), Na₂SO₃ (2 mM), Na₂S₂O₄ (2 mM), GSSG (glutathione disulfide, 2 mM), sulfonamide (2 mM), GSNO (2 mM), and NaHS (2 mM), respectively. 20 μL of the above solutions were transferred to a glass slide for the visualization with a fluorescence microscope with a 465/515 nm and an excitation/emission filter set.

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1]. Martelli A, Citi V,et,al. Vascular Effects of H2S-Donors: Fluorimetric Detection of H2S Generation and Ion Channel Activation in Human Aortic Smooth Muscle Cells. Methods Mol Biol. 2019;2007:79-87. doi: 10.1007/978-1-4939-9528-8_6. PMID: 31148107.

[2]. Li YJ, Chen J, et,al. In site bioimaging of hydrogen sulfide uncovers its pivotal role in regulating nitric oxide-induced lateral root formation. PLoS One. 2014 Feb 27;9(2):e90340. PMID: 24587333

产品描述

WSP-1 is a selective and rapid responsive fluorescent probe for active sulfides, specifically designed for the detection of hydrogen sulfide (H2S) within biological samples and intracellular environments. WSP-1 selectively and rapidly reacts with H2S, generating benzodithiolone and a fluorescent compound (Ex=465nm, Em=515nm)^[1].

WSP-1 (10 µM) can detect sulfide levels in RFL-6 cells where the sulfide donor GYY4137 (10-100 µM) is present[2]. The use of the WSP-1 fluorescent dye enables the detection of hydrogen sulfide (H2S) generation in human vascular smooth muscle cells following H2S donor administration[3]. WSP-1 can applied to track endogenous H2S in tomato (Solanum lycopersicum) roots in site[4].

References:
[1]. Liu C, Pan J, et,al. Capture and visualization of hydrogen sulfide by a fluorescent probe. Angew Chem Int Ed Engl. 2011 Oct 24;50(44):10327-9. doi: 10.1002/anie.201104305. Epub 2011 Sep 6. PMID: 21898737; PMCID: PMC3417056.
[2]. Cortese-Krott MM, Fernandez BO, et,al. Nitrosopersulfide (SSNO(-)) accounts for sustained NO bioactivity of S-nitrosothiols following reaction with sulfide. Redox Biol. 2014 Jan 11;2:234-44. doi: 10.1016/j.redox.2013.12.031. PMID: 24494198; PMCID: PMC3909780.
[3]. Martelli A, Citi V,et,al. Vascular Effects of H2S-Donors: Fluorimetric Detection of H2S Generation and Ion Channel Activation in Human Aortic Smooth Muscle Cells. Methods Mol Biol. 2019;2007:79-87. doi: 10.1007/978-1-4939-9528-8_6. PMID: 31148107.
[4]. Li YJ, Chen J, et,al. In site bioimaging of hydrogen sulfide uncovers its pivotal role in regulating nitric oxide-induced lateral root formation. PLoS One. 2014 Feb 27;9(2):e90340. doi: 10.1371/journal.pone.0090340. PMID: 24587333; PMCID: PMC3937356.

WSP-1是一种选择性和快速响应的活性硫化物荧光探针,专门用于检测生物样品和细胞内环境中的硫化氢(H2S)。WSP-1与H2S选择性快速反应,生成苯二噻吩酮和一种荧光化合物(Ex=465nm, Em=515nm)[1]。

WSP-1 (10 µM)可以检测含有硫化物供体GYY4137 (10-100 µM)的RFL-6细胞中的硫化物水平[2]。使用WSP-1荧光染料可以检测H2S供体给药后人血管平滑肌细胞中硫化氢(H2S)的生成[3]。WSP-1可用于现场跟踪番茄(Solanum lycopersicum)根系中的内源H2S[4]。

Chemical Properties

Cas No.	1352750-34-5	SDF
别名	荧光探针WSP-1
化学名	3'-methoxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6'-yl 2-(pyridin-2-yldisulfanyl)benzoate
Canonical SMILES	O=C(OC1=CC=C2C(OC3=C(C24C5=CC=CC=C5C(O4)=O)C=CC=C3)=C1)CO=C(OC1=CC=C2C(OC3=C(C24C5=CC=CC=C5C(O4)=O)C=CC(OC)=C3)=C1)C6=C(SSC7=NC=CC=C7)C=CC=C6
分子式	C₃₃H₂₁NO₆S₂	分子量	591.7
溶解度	2mg/mL in DMSO, 20mg/mL in DMF	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。
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。

溶解性数据

制备储备液
		1 mg	5 mg	10 mg
	1 mM	1.69 mL	8.4502 mL	16.9005 mL
	5 mM	0.338 mL	1.69 mL	3.3801 mL
	10 mM	0.169 mL	0.845 mL	1.69 mL

摩尔浓度计算器
稀释计算器
分子量计算器

计算

动物体内配方计算器 (澄清溶液)

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，注：如该浓度超过该批次药物DMSO溶解度，请先联系GLPBIO）；

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。

注意：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。

Research Update

Caenorhabditis elegans WSP-1 regulation of synaptic function at the neuromuscular junction

J Biol Chem 2010 Jul 23;285(30):23040-6.PMID:20501656DOI:10.1074/jbc.M109.096164.

The Rho GTPase members and their effector proteins, such as the Wiskott-Aldrich syndrome protein (WASP), play critical roles in regulating actin dynamics that affect cell motility, endocytosis, cell division, and transport. It is well established that Caenorhabditis elegans WSP-1 plays an essential role in embryonic development. We were interested in the role of the C. elegans protein WSP-1 in the adult nematode. In this report, we show that a deletion mutant of WSP-1 exhibits a strong sensitivity to the neuromuscular inhibitor aldicarb. Transgenic rescue experiments demonstrated that neuronal expression of WSP-1 rescued this phenotype and that it required a functional WSP-1 Cdc42/Rac interactive binding domain. WSP-1-GFP fusion protein was found localized presynaptically, immediately adjacent to the synaptic protein RAB-3. Strong genetic interactions with WSP-1 and other genes involved in different stages of synaptic transmission were observed as the WSP-1(gm324) mutation suppresses the aldicarb resistance seen in unc-13(e51), unc-11(e47), and snt-1 (md290) mutants. These results provide genetic and pharmacological evidence that WSP-1 plays an essential role to stabilize the actin cytoskeleton at the neuronal active zone of the neuromuscular junction to restrain synaptic vesicle release.

Caenorhabditis elegans WASP-interacting protein homologue WIP-1 is involved in morphogenesis through maintenance of WSP-1 protein levels

Biochem Biophys Res Commun 2006 Feb 10;340(2):709-17.PMID:16378591DOI:10.1016/j.bbrc.2005.12.056.

Mammalian WASP and N-WASP are involved in reorganization of the actin cytoskeleton through activation of the Arp2/3 complex and in regulation of cell motility or cell shape changes. In the present study, we identified WASP-interacting protein homologue (WIP)-1 in Caenorhabditis elegans. WIP-1 contains the domains and sequences conserved among mammalian WIP family proteins. Yeast two-hybrid analysis detected a physical interaction between WIP-1 and WSP-1, the sole homologue of WASP/N-WASP in C. elegans. Western analysis of embryo lysates showed that RNA interference (RNAi) treatment for wip-1 decreased levels of WSP-1 protein, and WSP-1(RNAi) treatment decreased levels of WIP-1 protein. However, WSP-1 mRNA levels were not decreased in wip-1(RNAi)-treated embryos, and wip-1 mRNA levels were not decreased in WSP-1(RNAi)-treated embryos. Furthermore, disruption of WIP-1 by RNAi resulted in embryonic lethality with morphologic defects in hypodermal cell migration, a process known as ventral enclosure. This phenotype was similar to that observed in RNAi experiments for WSP-1. Immunostaining showed that WIP-1 was expressed by migrating hypodermal cells, as was WSP-1. This expression during ventral enclosure was reduced in wip-1(RNAi)-treated embryos and WSP-1(RNAi)-treated embryos. Our results suggest that C. elegans WIP-1 may function in hypodermal cell migration during ventral enclosure by maintaining levels of WSP-1.

The H3K4me3/2 histone demethylase RBR-2 controls axon guidance by repressing the actin-remodeling gene WSP-1

Development 2016 Mar 1;143(5):851-63.PMID:26811384DOI:10.1242/dev.132985.

The dynamic regulation of histone modifications is important for modulating transcriptional programs during development. Aberrant H3K4 methylation is associated with neurological disorders, but how the levels and the recognition of this modification affect specific neuronal processes is unclear. Here, we show that RBR-2, the sole homolog of the KDM5 family of H3K4me3/2 demethylases in Caenorhabditis elegans, ensures correct axon guidance by controlling the expression of the actin regulator WSP-1. Loss of rbr-2 results in increased levels of H3K4me3 at the transcriptional start site of WSP-1, with concomitant higher WSP-1 expression responsible for defective axon guidance. In agreement, overexpression of WSP-1 mimics rbr-2 loss, and its depletion restores normal axon guidance in rbr-2 mutants. NURF-1, an H3K4me3-binding protein and member of the chromatin-remodeling complex NURF, is required for promoting aberrant WSP-1 transcription in rbr-2 mutants and its ablation restores wild-type expression of WSP-1 and axon guidance. Thus, our results establish a precise role for epigenetic regulation in neuronal development by demonstrating a functional link between RBR-2 activity, H3K4me3 levels, the NURF complex and the expression of WSP-1.

Caenorhabditis elegans Flamingo FMI-1 controls dendrite self-avoidance through F-actin assembly

Development 2020 Jul 31;147(14):dev179168.PMID:32631831DOI:10.1242/dev.179168.

Self-avoidance is a conserved mechanism that prevents crossover between sister dendrites from the same neuron, ensuring proper functioning of the neuronal circuits. Several adhesion molecules are known to be important for dendrite self-avoidance, but the underlying molecular mechanisms are incompletely defined. Here, we show that FMI-1/Flamingo, an atypical cadherin, is required autonomously for self-avoidance in the multidendritic PVD neuron of Caenorhabditis elegans The fmi-1 mutant shows increased crossover between sister PVD dendrites. Our genetic analysis suggests that FMI-1 promotes transient F-actin assembly at the tips of contacting sister dendrites to facilitate their efficient retraction during self-avoidance events, probably by interacting with WSP-1/N-WASP. Mutations of vang-1, which encodes the planar cell polarity protein Vangl2 previously shown to inhibit F-actin assembly, suppress self-avoidance defects of the fmi-1 mutant. FMI-1 downregulates VANG-1 levels probably through forming protein complexes. Our study identifies molecular links between Flamingo and the F-actin cytoskeleton that facilitate efficient dendrite self-avoidance.

WIP-1 and DBN-1 promote scission of endocytic vesicles by bridging actin and Dynamin-1 in the C. elegans intestine

J Cell Sci 2019 Jun 17;132(12):jcs228023.PMID:31118234DOI:10.1242/jcs.228023.

There has been a consensus that actin plays an important role in scission of the clathrin-coated pits (CCPs) together with large GTPases of the dynamin family in metazoan cells. However, the recruitment, regulation and functional interdependence of actin and dynamin during this process remain inadequately understood. Here, based on small-scale screening and in vivo live-imaging techniques, we identified a novel set of molecules underlying CCP scission in the multicellular organism Caenorhabditis elegans We found that loss of Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP-1) impaired CCP scission in a manner that is independent of the C. elegans homolog of WASP/N-WASP (WSP-1) and is mediated by direct binding to G-actin. Moreover, the cortactin-binding domain of WIP-1 serves as the binding interface for DBN-1 (also known in other organisms as Abp1), another actin-binding protein. We demonstrate that the interaction between DBN-1 and F-actin is essential for Dynamin-1 (DYN-1) recruitment at endocytic sites. In addition, the recycling regulator RME-1, a homolog of mammalian Eps15 homology (EH) domain-containing proteins, is increasingly recruited at the arrested endocytic intermediates induced by F-actin loss or DYN-1 inactivation, which further stabilizes the tubular endocytic intermediates. Our study provides new insights into the molecular network underlying F-actin participation in the scission of CCPs.This article has an associated First Person interview with the first author of the paper.

WSP-1

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