
- 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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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.
2.尿素:有膜蛋白的损失
3.RIPA:用于磷酸化蛋白的温和裂解,不破坏磷酸基团
浙江大学奚振宇、徐又一、朱利平等2009年在journal of membrane science(中文翻译为《膜科学杂志》)发表的一篇论文,其中探讨了缓冲溶液的pH值对聚乙烯-多巴胺复合膜亲水性的影响,发现缓冲溶液的pH值为8.5时制备的复合膜亲水性最强。
通过大量的实验,研究人员发现,pH值为8.5时制备的多巴胺复合膜性能最优。这个是通过大量的实验总结得到的结论。

