FAM-VAD-FMK

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Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	691.66
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.32
Correction Factor (280 nm)	0.178
Extinction coefficient (cm ^-1 M ^-1)	83000
Excitation (nm)	493
Emission (nm)	517

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

Molecular weight

691.66

Correction Factor (260 nm)

0.32

Correction Factor (280 nm)

0.178

Extinction coefficient (cm ^-1 M ^-1)

83000

Excitation (nm)

493

Emission (nm)

517

FAM-VAD is a green 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 FAM-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. It works in diverse cell lines such as human, rodent and drosophila.

Example protocol

AT A GLANCE

Important It is important to store at <-15 °C and should be stored in cool, dark place.
Following protocol only provides a guideline, and should be modified according to your specific needs.
It can be used within 12 months from the date of receipt.

SAMPLE EXPERIMENTAL PROTOCOL

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

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 in Hanks with 20 mM Hepes buffer (HHBS).
Mix equal volume of the treated cells with 2X caspase substrate assay solution, and incubate the cells in a 37 °C, 5% CO₂ incubator for at least 1 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# 13484, 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 FAM-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	144.58 µL	722.899 µL	1.446 mL	7.229 mL	14.458 mL
5 mM	28.916 µL	144.58 µL	289.159 µL	1.446 mL	2.892 mL
10 mM	14.458 µL	72.29 µL	144.58 µL	722.899 µL	1.446 mL

Molarity calculator

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Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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Spectral properties

Correction Factor (260 nm)	0.32
Correction Factor (280 nm)	0.178
Extinction coefficient (cm ^-1 M ^-1)	83000
Excitation (nm)	493
Emission (nm)	517

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Correction Factor (280 nm)
TF4-VAD-FMK	578	602	90000	0.436
SRB-VAD-FMK [Sulforhodamine B-VAD-FMK]	559	577	-	-
FAM-LETD-FMK	493	517	83000	0.178

Images

Figure 1. Chemical structure for FAM-VAD-FMK

Citations

View all 1 citations: Citation Explorer

Heat-killed Mycobacterium tuberculosis prime-boost vaccination induces myeloid-derived suppressor cells with spleen dendritic cell--killing capability
Authors: Ribechini, Eliana and Eckert, Ina and Beilhack, Andreas and Du Plessis, Nelita and Walzl, Gerhard and Schleicher, Ulrike and Ritter, Uwe and Lutz, Manfred B
Journal: JCI insight (2019)

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)

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

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

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

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

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

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