
- 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 |
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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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因为常用的缓冲溶液由弱酸及其共轭酸盐组合而成,当加入酸时,弱酸盐部分转化为弱酸;当加入碱时,弱酸部分转化为弱酸盐。而弱酸在水中不完全电离,弱酸盐在水中可以水解,所以ph变化较小,保持了溶液的PH基本不变。
百度教育团队【海纳百川团】为您解答。
感谢您的采纳 O(∩_∩)O 。如有疑问,欢迎追问。
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。
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。
甲液:0.05mol/L Na2HPO4溶液 :称取磷酸氢二钠9.465g 7.099g,加蒸 馏水至1000ml ;
乙液:0.05mol/L KH2P04溶液: 称取磷酸二氢钾, 9.07g 6.803g,加蒸馏水至1000m1.
将甲乙液分装在棕色瓶内,于4℃冰箱中保存,用时甲、乙两液各按不同比例混合,即可得所需pH的缓冲液,见下表:
pH 甲液ml 乙液mI
5.29 2.5 97.5
5.59 5.0 95.0
5.91 10.0 90.0
6.24 20.0 80.0
6.47 30.0 70.0
6.64 40.0 60.0
6.81 50.0 50.0
6.98 60.0 40.0
7.17 70.0 30.0
7.38 80.0 20.0
7.73 90.0 10.0
8.04 95.0 5.0
你要配制pH=6.8的PBS,可用甲、乙液各50ml混合即可.

