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SRB-VAD-FMK [Sulforhodamine B-VAD-FMK]
SRB-VAD is a red fluorescent cell-permeable polycaspase inhibitor to target caspases 1, 2, 3, 6, 8, 9, or 10. Once inside the cell, the inhibitor binds covalently to the active caspase, which produces a green fluorescence. When added to a population of cells, the SRB-VAD-FMK probe enters each cell and covalently binds to a reactive cysteine residue that resides on the large subunit of the active caspase heterodimer, thereby inhibiting further enzymatic activity. The bound labeled reagent is retained within the cell, while any unbound reagent will diffuse out of the cell and is washed away. The green fluorescent signal is a direct measure of the amount of active caspase present in the cell at the time the reagent was added. Cells that contain the bound labeled reagent can be analyzed by 96-well plate-based fluorometry, fluorescence microscopy, or flow cytometry. The probe has the spectral properties similar to Cy3® and TRITC, thus the filter set of Cy3® and TRITC can be conveniently used with a flow cytometer or microscope.
Example protocol
AT A GLANCE
Important It is important to store at <-15 °C and should be stored in cool, dark place.
It can be used within 12 months from the date of receipt.
It can be used within 12 months from the date of receipt.
SAMPLE EXPERIMENTAL PROTOCOL
Following protocol only provides a guideline, and should be modified according to your specific needs.
- Prepare a 10 mM stock solution in DMSO.
- Prepare a 2X caspase substrate (50 µM) assay solution as the following: 50 µL substrate stock solution, 100 µL DTT (1M), 400 µL EDTA (100 mM), 10 mL Tris Buffer (20 mM), pH =7.4.
- Mix equal volume of the caspase standards or samples with 2X caspase substrate assay solution, and incubate the solutions at room temperature for at least 1 hour.
- Monitor the fluorescence using a fluorescence microplate reader, or absorbance using an absorbance microplate reader.
Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes
- Prepare a 2-5 mM stock solution in DMSO.
- Treat cells as desired.
- Prepare a 2X permeable caspase substrate (20 µM) assay solution by diluting the DMSO stock solution (from Step 2.1) in Hanks with 20 mM Hepes buffer (HHBS).
- Mix equal volume of the treated cells with 2X caspase substrate assay solution (from Step 2.3), and incubate the cells in a 37°C, 5% CO2 incubator for at least1 hour.
- Wash the cells with HHBS for at least once.
- Monitor the fluorescence intensity by a flow cytometer, a fluorescence microscope or a fluorescence microplate reader.
Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes (For #13470-13476 only)
- Prepare a 250X stock solution by adding 50 µL DMSO into the vial.
Note For Cat# 13482, prepare a 250X stock solution by adding 200 µL DMSO into the vial. - Treat cells as desired.
- Add 250 X DMSO stock solution into the cell solution at a 1:250 ratio (such as 2 µL to 500 µL cells), and incubate the cells in a 37°C, 5% CO2 incubator for 1 hour.
- Wash the cells with HHBS for at least once.
- Monitor the fluorescence intensity by flow cytometer, fluorescence microscopy or fluorescent microplate reader.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of SRB-VAD-FMK [Sulforhodamine B-VAD-FMK] to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 114.415 µL | 572.076 µL | 1.144 mL | 5.721 mL | 11.442 mL |
| 5 mM | 22.883 µL | 114.415 µL | 228.83 µL | 1.144 mL | 2.288 mL |
| 10 mM | 11.442 µL | 57.208 µL | 114.415 µL | 572.076 µL | 1.144 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Correction Factor (280 nm) |
| FAM-VAD-FMK | 493 | 517 | 83000 | 0.178 |
| TF4-VAD-FMK | 577 | 602 | 90000 | 0.436 |
Citations
View all 2 citations: Citation Explorer
TRPV4-Rho signaling drives cytoskeletal and focal adhesion remodeling in trabecular meshwork cells
Authors: Lakk, Monika and Kri{\v{z}}aj, David
Journal: American Journal of Physiology-Cell Physiology (2021)
Authors: Lakk, Monika and Kri{\v{z}}aj, David
Journal: American Journal of Physiology-Cell Physiology (2021)
Mechanically induced cytoskeletal remodeling in trabecular meshwork cells requires TRPV4-Rho signaling interactions
Authors: Lakk, Monika and Kri{\v{z}}aj, David
Journal: bioRxiv (2020)
Authors: Lakk, Monika and Kri{\v{z}}aj, David
Journal: bioRxiv (2020)
References
View all 26 references: Citation Explorer
Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties
Authors: Lawrence CP, Chow SC.
Journal: Toxicol Appl Pharmacol. (2012)
Authors: Lawrence CP, Chow SC.
Journal: Toxicol Appl Pharmacol. (2012)
Inhibition of elicitation of allergic contact dermatitis by topical use of Z-VAD-FMK, a broad caspase inhibitor: experiment in mice
Authors: Li YY, Yan CL.
Journal: Zhonghua Yi Xue Za Zhi (2012): 1992
Authors: Li YY, Yan CL.
Journal: Zhonghua Yi Xue Za Zhi (2012): 1992
Structure of human caspase-6 in complex with Z-VAD-FMK: New peptide binding mode observed for the non-canonical caspase conformation
Authors: Muller I, Lamers MB, Ritchie AJ, Dominguez C, Munoz-Sanjuan I, Kiselyov A.
Journal: Bioorg Med Chem Lett (2011): 5244
Authors: Muller I, Lamers MB, Ritchie AJ, Dominguez C, Munoz-Sanjuan I, Kiselyov A.
Journal: Bioorg Med Chem Lett (2011): 5244
Intracochlear perfusion of leupeptin and z-VAD-FMK: influence of antiapoptotic agents on gunshot-induced hearing loss
Authors: Abaamrane L, Raffin F, Schmerber S, Sendowski I.
Journal: Eur Arch Otorhinolaryngol (2011): 987
Authors: Abaamrane L, Raffin F, Schmerber S, Sendowski I.
Journal: Eur Arch Otorhinolaryngol (2011): 987
Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death
Authors: Meslin B, Beavogui AH, Fasel N, Picot S.
Journal: PLoS One (2011): e23867
Authors: Meslin B, Beavogui AH, Fasel N, Picot S.
Journal: PLoS One (2011): e23867
Page updated on November 21, 2024
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