- Description
- Additional Information
- Readable Documents
- Assay Principle
- Reviews
Key Benefits
- Simultaneous detection of mitochondrial membrane potential and caspase activity.
- Readout – Flow cytometry, Fluorescent plate reader, Fluorescent microscopy .
- Reliable: Yields both quantitative and qualitative results. Gives a strong positive signal.
- The kit can be used in conjunction with other antibodies or stains.
- Ease Of Use: No need to make cell lysates or run western blots.
- Cell Permeable Reagents.
Additional information
Kit Size | 25, 100 |
---|---|
Caspase | Poly Caspase, Caspase 3/7, Caspase 8, Caspase 9, Caspase 1 |
Caspase (poly, 3/7, 8, 9, 1) & Mitochondria Membrane Potential Detection – MitoCasp
Caspase enzymes specifically recognize a 4 amino acid sequence (on their substrate) which necessarily includes an aspartic acid residue. This residue is the target for the cleavage reaction, which occurs at the carbonyl end of the aspartic acid residue(6). Caspases can be detected via immunoprecipitation, immuno-blotting techniques using caspase specific antibodies, or by employing fluorogenic substrates which become fluorescent upon cleavage by the caspase. MitoCasp uses a novel approach to detect active caspases (7-9). The methodology is based on carboxyfluorescein (FAM) labeled fluoromethyl ketone (FMK)-peptide inhibitors of caspases. These inhibitors are cell permeable and non-cytotoxic. Once inside the cell, the inhibitor binds covalently to the active caspase (10). Cells that contain bound inhibitor can be analyzed by flow cytometry or fluorescence microscopy.
Cell Technology utilizes a cationic dye to visualize mitochondrial membrane potential (15-17). The cationic dye is cell permeable and has a strong fluorescent signal in the red region and exhibits low membrane potential independent (non specific) binding and toxicity. In healthy cells the cationic dye is accumulated by the mitochondria in proportion to the DeltaPsi (membrane potential). In most cell lines, accumulation of the cationic dye in the mitochondria results in a higher fluorescence intensity. In apoptotic cells, where the mitochondrial membrane potential is compromised, the cationic dye does not accumulated in the mitochondria and these cells exhibit a lower fluorescence signal. Utilizing these two reagents in combination Caspase activity and mitochondrial membrane potential can be analyzed simultaneously. Citations Identification of single-domain, Bax-spec
Jurkat cells were stimulated with Staurosporine for 3 hours (B) or DMSO (A). The cells were then stained with the MitoCasp kit according to the protocol. The cells were then washed twice and analyzed by flow cytometry: Ex:488nm Em: FL1 and FL2.
Fig A. Healthy cells show a strong red fluorescence indicating intact mitochondria and no green fluorescence, indicating no active caspases.
Fig B. Apoptotic cells show a loss of red fluorescence (y axis) indicating loss of mitochondrial membrane potential and positive green fluorescence (x axis) indicating active caspases.
Document Title |
MitoCaspProtocol |
MitoCasp Datasheet |
msds.MitoCasp |
Title | File | Link | Author(s) | Journal | Year; Edition:Pages |
Selective cytotoxicity of Pancratistatin-related natural Amaryllidaceae alkaloids: evaluation of the activity of two new compounds | http://www.cancerci.com/content/7/1/10 | Griffin, Sharda, Sood, Nair, et al | Cancer Cell Int | V7 2007 | |
OSU-03012, a Novel Celecoxib Derivative, Is Cytotoxic to Myeloma Cells and Acts through Multiple Mechanisms | http://clincancerres.aacrjournals.org/cgi/content/abstract/13/16/4750 | Zhang, Suvannasankha, Crean, et.al | Clinical Cancer Research | 13, pp 4750-4758, Aug 15, 2007 | |
Galectin-3 but not galectin-1 induces mast cell death by oxidative stress and mitochondrial permeability transition | http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T20-4RTM2SR-2&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=ed18c5ccefa05603a620115964776eea | Yoshihiro Suzuki, Toshio Inoue, Tetsuro Yoshimaru and Chisei Ra | Biochimica et Biophysica Acta (BBA) - Molecular Cell Research | Vol 1783, issue 5, pp 924-934, 2008 | |
Nitric oxide protects mast cells from activation-induced cell death: the role of the phosphatidylinositol-3 kinase-Akt-endothelial nitric oxide synthase pathway | http://www.jleukbio.org/content/83/5/1218.short | Toshio Inoue, Yoshihiro Suzuki, Tetsuro Yoshimaru and Chisei Ra | Journal of Leukocyte Biology | vol. 83 no. 5, pp1218-1229, 2008 doi:10.1189/jlb.1007667 |
Reference |
Slee, E. A., C. Adrain, and S. J. Martin. 1999. Serial Killers: ordering caspase activation events in apoptosis. Cell Death and Differ. 6:1067-1074. |
Walker, N. P., R. V. Talanian, K. D. Brady, L. C. Dang, N. J. Bump, C. R. Ferenz, S. Franklin, T. Ghayur, M. C. Hackett and L. D. Hammill. 1994. Crystal Structure of the Cysteine Protease Interleukin-1ß-Converting Enzyme: A (p20/p10)2 Homodimer. Cell 78:343-352. |
Wilson, K. P., J. F. Black, J. A. Thomson, E. E. Kim, J. P. Griffith, M. A. Navia, M. A. Murcko, S. P. Chambers, R. A. Aldape, S. A. Raybuck, and D. J. Livingston. 1994. Structure and mechanism of interleukin-1 beta converting enzyme. Nature 370: 270-275. |
Rotonda, J., D. W. Nicholson, K. M. Fazil, M. Gallant, Y. Gareau, M. Labelle, E. P. Peterson, D. M. Rasper, R. Ruel, J. P. Vaillancourt, N. A. Thornberry and J. W. Becker. 1996. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nature Struct. Biol. 3(7): 619-625. |
Kumar, S. 1999. Mechanisms mediating caspase activation in cell death. Cell Death and Differ. 6: 1060-1066. |
Thornberry, N. A., T. A. Rano, E. P. Peterson, D. M. Rasper, T. Timkey, M. Garcia-Calvo, V. M. Houtszager, P. A. Nordstrom, S. Roy, J. P. Vaillancourt, K. T. Chapman and D. W. Nicholson. 1997. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272(29): 17907-17911. |
Amstad, P.A., G.L. Johnson, B.W. Lee and S. Dhawan. 2000. An in situ marker for the detection of activated caspases. Biotechnology Laboratory 18: 52-56. |
Bedner, E., P. Smolewski, P.A. Amstad and Z. Darzynkiewicz. 2000. Activation of caspases measured in situ by binding or fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Research 259: 308-313. |
Smolewski, P., E. Bedner, L. Du, T.-C. Hsieh, J. Wu, J. D. Phelps and Z. Darzynkiewicz. 2001. Detection of caspase activation by fluorochrome-labeled inhibitors: multiparameter analysis by laser scanning cytometry. Cytometry 44: 73-82. |
Ekert, P. G., J. Silke and D. L. Vaux. 1999. Caspase inhibitors. Cell Death and Differ. 6:1081-1086. |
Desagher, S., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B., and Martinou, J.C. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144 (5): 891-901 (1999). |
Narita, M., Shimizu, S., Ito, T., Chittenden, T., Lutz, R. J., Matsuda, H., and Tsujimoto, Y. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc. Natl. Acad. Sci. USA 95: 14681-14686 (1998). |
Basanez, G., Nechushtan, A., Drozhinin, O., Chanturiya, A., Choe, E., Tutt, S., Wood, K. A., Hsu, Y. T., Zimmerberg, J., and Youle, R. J. Bax , but not Bcl-XL decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc. Natl. Acad. Sci. USA 96: 5492-5497 (1999). |
Luo, X., Budihardio, I., Zou, H., Slaughter, C., and Wang, X. Bid, a Bcl-2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481-490 (1998). |
Ehrenberg B, Montana V, Wei MD, Wuskell JP, Loew LM. Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes. Biophys J. 1988 May;53(5):785-94. |
Farkas DL, Wei MD, Febbroriello P, Carson JH, Loew LM. Simultaneous imaging of cell and mitochondrial membrane potentials. : Biophys J. 1989 Dec;56(6):1053-69. Erratum in: Biophys J 1990 Mar;57(3):following 684. |
Russell C. Scaduto, Jr. and Lee W. Grotyohann. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J. 1999 Jan;76(1 Pt 1):469-77. |
Rajagopal A, Pant AC, Simon SM, Chen Y. In vivo analysis of human multidrug resistance protein 1 (MRP1) activity using transient expression of fluorescently tagged MRP1. Cancer Res. 2002 Jan 15;62(2):391-6. |
Part# | Reagent | Temperature |
Part # 4015 | Mitochondrial Memberane Potential Cationic Dye | 2-8C |
Refer to Product Datasheet | Caspase Detection Reagent (Poly Caspase, Caspase 3/7, Caspase 1, Caspase 8 or Caspase 9) | 2-8C |
Part # 3028 | 10X Wash Buffer | 2-8C |
Part # 3032 | 1X Dilution Buffer | 2-8C |
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制冰机原理:
制冰机是一种将水通过蒸发器由制冷系统制冷剂冷却后生成冰的制冷机械设备。根据蒸发器和生成过程方式原理不同,生成的冰的形状也不同,人们一般根据冰形状将制冰机分为颗粒冰机、片冰机、板冰机、管冰机、壳冰机等等。
制冰机的分类:
按冰的形状可分:鳞形片冰机,雪花制冰机,条冰机,板冰机,块冰机(可分食用小块冰机和工业用大块冰机),冰粒机,颗粒机,管冰机,子弹头制冰机。
实验室制冰机种类选择:
我们平时在外面就餐时,经常看到商家在饮料中加入冰块,有时候是方块的,有时候是中空的,还有在菜市场买海鲜的时候也会看到下面铺着一层冰,究竟有什么区别吗?我们在开展实验室工作的时候,应如何选择制冰机呢?
实验室制冰机主要是用于在开展生命科学相关实验时,给样品提供冰浴环境,防止样品(如核酸、蛋白)因为温度的变化导致降解或变质,从而保障实验结果的准确性和有效性。绝大部分的时候,我们的样品是放在EP管、离心管、pcr管等容器里面,然后把样品管插在冰浴中,靠接触来实现温度的保持。由于样品和冰无法直接接触,而是要通过管壁来传输,所以管壁和冰浴的接触面积是冰浴效果的保障,为了更好的保障样品,应尽量的扩大管壁和冰浴的接触面积。
针对以上考虑,实验室制冰机一般选择雪花制冰机,该种类型的制冰机生产出来的冰呈雪花状碎冰,由于颗粒很小,可以和管壁进行密切的接触,保障冰浴效果。
对于出冰的具体要求中,还有一点是非常重要的,出来的雪花冰要求有15-25%的含水量,这样可极大的提高冰浴和管壁的接触效果。
文章来源:厦门国仪科学仪器有限公司
因为本实验室要建立基因的分离、克隆、表达的平台,需要以下设备,请内行人给个大体的报价,谢谢。我的信箱:zju882003@yahoo.com.cn
此过程如下:PCR扩增目的基因→T-A克隆,酶切鉴定,测序→亚克隆构建表达载体,酶切鉴定,测序→IPTG诱导重组融合蛋白表达→SDS-PAGE检测重组融合蛋白分子量大小→NTA亲和层析法纯化重组融合蛋白→WesternBlot检测融合蛋白免疫反应
1.生化细菌培养箱
2.制冰机
3.PH仪
4.电转化仪(要好的公司提供的产品)
5.蒸馏器
6.高压消毒锅
7.夹心式垂直电泳槽(要好的公司提供的产品)
8.稳压稳流电泳仪
9.磁力搅拌器
10.细菌摇床
11.超声细菌破碎仪
14.转膜仪(要好的产品)
15.融合蛋白的纯化系统(要最好的产品)
16.4℃冰箱
17.微量移液器(1-10ul,10-100ul,100-1000ul)Eppendorf
18.普通照相机
19.紫外分光光度计
20.超净工作台
不过制冰机里的冰不可以,那冰怎么得到呢?
protocol上面说在离心菌液的时候离心管里面要有1/2的冰啊,这个冰应该是碎冰而不是直接冻出来的整块的冰吧。
大家是怎么得到的呢?谢谢!!
谢谢各位!
概括了以下四点:
1.在用完制冰机后需要将冰箱内的冰块清理干净。清洗制冰机时应关掉电源,不要用水管直接对准机身冲洗,用清水,加入适量中性洗涤剂(严禁用酸性、碱性等腐蚀性溶剂)擦拭机器围板和内胆,再用软布擦干净。
2.制冰机必须两个月旋开进水软管管头,清洗进水阀滤网,避免水中砂泥杂质堵塞进水口,而引起进水量变小,导致不制冰。
3.每二个月清扫冷凝器表面灰尘,冷凝散热不良会引起压缩机部件损坏。清扫时,使用吸尘器、小毛刷等清洗冷凝表面油尘,不能使用尖锐金属工具清扫,以免损坏冷凝器。
4.制冰机的水管、水槽、储冰箱及保护胶片要每两个月清洗一次。不使用时,应清洗干净,并用电吹风吹干冰模及箱内水分,放在无腐蚀气体及通风干燥的地方,避免露天存放。