TF4-VAD-FMK

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Chemical structure for TF4-VAD-FMK
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25 Tests 13471 $95


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Overview

Ex/Em (nm)588/610
MW878.01
SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
Category Enzyme Detection
Peptidases and Proteases
Related Cell Apoptosis
Apoptosis and Cytotoxicity
TF4-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 TF4-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 Texas Red®, thus the filter set of Texas Red® can be conveniently used with a flow cytometer or microscope.




Calculators
Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of TF4-VAD-FMK to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.



Molarity calculator

Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =
 






Spectrum Advanced Spectrum Viewer

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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.

1.       General Solution Caspase Assays Using AMC, AFC, pNA, R110 and ProRed Substrates

1.1.     Prepare a 10 mM stock solution in DMSO.

1.2.     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

1.3.     Mix equal volume of the caspase standards or samples with 2X caspase substrate assay solution (from Step 1.2), and incubate the solutions at room temperature for at least 1 hour.

1.4.     Monitor the fluorescence using a fluorescence microplate reader, or absorbance using an absorbance microplate reader.

2.       Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes

2.1.     Prepare a 2-5 mM stock solution in DMSO.

2.2.     Treat cells as desired.

2.3.     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).

2.4.     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.

2.5.     Wash the cells with HHBS for at least once.

2.6.     Monitor the fluorescence intensity by a flow cytometer, a fluorescence microscope or a fluorescence microplate reader.

3.       Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes (For #13470-13476 only)

3.1.     Prepare a 250X stock solution by adding 50 µL DMSO into the vial.

3.2.     Treat cells as desired.

3.3.     Add 250 X DMSO stock solution (from Step 3.1) into the cell solution at a 1:250 ratio (such as 2 uL to 500 uL cells), and incubate the cells in a 37°C, 5% CO2 incubator for 1 hour.

3.4.     Wash the cells with HHBS for at least once.

3.5.     Monitor the fluorescence intensity by flow cytometer, fluorescence microscopy or fluorescent microplate reader.






References & Citations

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)

[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

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

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

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

Pretreatment with pancaspase inhibitor (Z-VAD-FMK) delays but does not prevent intraperitoneal heat-killed group B Streptococcus-induced preterm delivery in a pregnant mouse model
Authors: Equils O, Moffatt-Blue C, Ishikawa TO, Simmons CF, Ilievski V, Hirsch E.
Journal: Infect Dis Obstet Gynecol (2009): 749432

[Attenuation of allergic contact dermatitis by Z-VAD-FMK, a broad caspase inhibitor: experiment with mice]
Authors: Li YY, Yan CL, Xu W.
Journal: Zhonghua Yi Xue Za Zhi (2008): 3153

Bodipy-VAD-Fmk, a useful tool to study yeast peptide N-glycanase activity
Authors: Witte MD, Descals CV, de Lavoir SV, Florea BI, van der Marel GA, Overkleeft HS.
Journal: Org Biomol Chem (2007): 3690

Caspase inhibitor Z-VAD-FMK enhances the freeze-thaw survival rate of human embryonic stem cells
Authors: Heng BC, Clement MV, Cao T.
Journal: Biosci Rep (2007): 257

Effects of caspase inhibitors (z-VAD-fmk, z-VDVAD-fmk) on Nile Red fluorescence pattern in 7-ketocholesterol-treated cells: investigation by flow cytometry and spectral imaging microscopy
Authors: Vejux A, Lizard G, Tourneur Y, Riedinger JM, Frouin F, Kahn E.
Journal: Cytometry A (2007): 550


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Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Enzyme Probes & Assay Kits
2. Cell Apoptosis & Proliferation

Certificate of Analysis