- Description
- Additional Information
- Readable Documents
- Assay Principle
- Reviews
Key Benefits
- Non-cytotoxic assay arrests further apoptotic activity via caspase inhibition.
- Cell permeablity permits direct visualization of cytosolic apoptotic events.
- Apoptotic cell population does not diminish over time.
- Add reagent directly to cells. No special buffer or media needed. No preparation of cell lysates required. Simple wash procedure.
- Works in diverse cell lines: human, rodent, Drosophila.
- Can be performed in conjunction with Annexin staining, TUNEL, antibody staining, or with other APO LOGIX reagents on the same population of cells.
- Permits high through-put screening. Protocol can be adapted for ex vivo as well as in situ experiments.
- Applications – Works with fluorescence microscope, 96-well fluorescence plate readers
- Yields both quantitative and qualitative results. Gives strong signal with little background noise.
Additional information
| Kit Size | 25, 100 |
|---|
APO LOGIX SR kits contain a generic sulforhodamine labeled caspase inhibitor (sulforhodamine-peptide-fluoromethyl ketone). This reagent is cell permeable and is used on whole cells to detect apoptosis. Apoptotic cells are detected by a fluorescence plate reader or fluorescence microscope using an excitation source at 550nm and measuring emission at 595nm. The assay takes about 1 hr to completeAPO LOGIX Sulforhodamine
Jurkat cells stimulated with staurosporine for 2 hours and then labeled with SR-VAD-FMK.
Left side: 30X phase contrast
Right side: 30X fluorescence microscope. Excitation: 550nm emission > 580nm.APO LOGIX Sulforhodamine
Jurkat cells stimulated with staurosporine for 2 hours. Cells were then stained with SR-VAD-FMK for 1 hour and read in a 96 well fluorescence plate reader.
| Document Title |
| SR protocol |
| SRVADFMK Datasheet |
| msds.Apologix |
| 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 SRily 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. |
| Carcia-Calvo, M., E. Peterson, B. Leiting, R. Ruel, D. Nicholson and N. Thornberry. 1998. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 273: 32608-32613. |
| Hirata, H., A. Takahashi, S. Kobayashi, S. Yonehara, H. Sawai, T. Okazaki, K. Yamamoto and M. Sasada. 1998. Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J. Exp. Med. 187: 587-600 |
| Part# | Reagent | Temperature |
| Part # 679 | Lyophilized SR-VAD-FMK | 2-8C |
| Part # 635 | 10X Wash Buffer | 2-8C |
| Part # 636 | 10X Fixitive | 2-8C |
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1、根据所需求的酸碱性选择合适的缓冲对,若是配制酸性缓冲液就选择弱酸与弱酸盐缓冲对;若是配制碱性缓冲液就选择弱碱与弱碱盐缓冲对。
2、根据所需要控制的PH范围以及弱酸和弱碱的解离常数(pKa/pKb)选择具体的共轭酸碱对,公式为PH=pKa±1=(14-pKb)±1。
3、根据公式计算缓冲溶液的组分比,酸性缓冲液PH=pKa-lgc酸/c盐,碱性缓冲液PH=14-pKb+lgc碱/c盐。
4、根据共轭酸碱对以及其组分比配制缓冲液,方法同普通溶液。
举例:试配制一种缓冲液,体积为1L,PH能维持在10.25左右。
a、依题意选择弱碱与弱碱盐的共轭对;
b、由PH=(14-pKb)±1,算出pKb在3.75与4.75之间,查弱碱的解离常数表可知氨水(pKb=4.75)符合要求,故可选择NH3-NH4Cl体系;
c、由PH=14-pKb+lgc碱/c盐,算出c(氨水)/c(氯化铵)=10;
d、设氨水的浓度为10mol/L,则氯化铵的浓度为1mol/L,所以在浓度为10mol/L体积为1L的氨水中加入1mol的氯化铵即可。
酶提取技术,属地球化学勘查学科。其是由克拉克(J.R.Clark)等人于20世纪80年代末和90年代初研制出的一种利用葡萄糖氧化酶提取矿物颗粒表面的非晶质锰的氧化膜寻找隐伏矿的方法。1995年以后已广泛应用。
根据组成不同,可分为两种,弱酸及其对应的强碱弱酸盐,弱碱及其对应的强酸弱碱盐。
因为HF可以和NaOH反应生成NaF和水,当NaOH反应完之后,NaF就可以与HF组成缓冲溶液,所以说可以直接使用NaOH和HF来配制缓冲溶液。
这也是一般配制缓冲溶液的方法,也就是用强碱和弱酸(或者强酸和弱碱)来配制缓冲溶液。

