DNs-Rh (Cell-based GST Activity Assay Reagent)

Product#: FDV-0030
$300.00
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DNs-Rh (Cell-based GST Activity Assay Reagent)

Cat.No. FDV-0030
Size 0.1 µmol 
Storage  -20ºC

Description

Novel activity assay probe for GSTs in live cells DNs-Rh < Cell-based GST Activity Assay Reagent >
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Glutathione S-Transferases (GSTs) are widely conserved in nature from bacteria to plants and animals. In human, over 20 members are identified and classified into three subfamilies. GSTs play an important role in detoxification of endogenous toxic metabolism or xenobiotics through converting them to glutathione (GSH)-conjugates.
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As many studies suggested, expression level of GSTs is significantly increased in cancer cells, GSTs are considered as anti-cancer drug-resistant enzymes in malignant cancer cells through the neutralization of drugs. To understand biological functions of GSTs, tools for monitoring GST activity are very important. Although several reagents including CDNB (conventional GSTs probe) for this purpose have been developed, no tool to measure intracellular GST activity was commercially available.

DNs-Rh is a novel fluorogenic substrate of GSTs discovered by Dr. Hiroshi Abe, Nagoya University. DNs-Rh is a rhodamine 110 derivative which protected by DNs (dinitrobenzenesulfonamide). This probe emits very low fluorescence (quantum yield = 0.0007) at normal state. After deprotected by thiols, rhodamine 110 is released and exhibits strong fluorescence (quantum yield = 0.645, S/N ratio ~900). DNs-Rh can be used as a fluorogenic GST activity assay probe. An important advantage of this probe is high cell-permeability and this probe is applied to intracellular GST activity under live cell condition. As DNs group is a well characterized substrate for various types of GST members, DNs-Rh is able to monitor pan-GST activity both in cell and in vitro.

Various applications are available. DNs-Rh is a powerful tool not only to investigate GST activity in live cell upon any biological stimulation, but also to develop GST inhibitors under live cell condition. This product has been commercialized under the license from Nagoya University.

Conventional method vs. DNs-Rh

Probe

 

Conventional method (CDNB)

DNs-Rh

Measuring Method

 

UV

Green Fluorescence

Application

in vitro

Possible

Possible

Live Cell Imaging

No

Yes

Flow Cytometry

No

Yes

Sensitivity

 

Low

High

Throughput

 

Low

High

Speficity

 

Low

High

Co-staining

 

No

Yes


Chemical Information
  • Formulation :C32H18N6O15S2
  • CAS No. : 3829-23-0
  • Molecular weight :790.6 g/mol
  • Solubility : Soluble in DMSO
  • Ex/Em: 496/520 nm (Rhodamine 110)
*Commercial FITC filter sets are available.

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Application
Intracellular GST activity assay in live cells (Imaging or flow cytometry)
In vitro GST activity assay

 
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Fig.1 Fluorescent spectrum
Fluorescent spectrum of DNs-Rhexcited at 490 nm both in the presence and absence of GST/GSH was measured.

Only in the GST/GSH, strong fluorescence around 520 nm was observed. DNs-Rh; 1 μM, GST; human recombinant GSTP1-1 10 μg/ml, GSH; 1 mM.
 


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Fig.2 in vitro GST activity assay
Assay solution containing 1 μM DNs-Rh, 1 mM GSH and 10 μg/ml recombinant human GSTP1-1 was incubated at 37℃ for 30 min.

DNs-Rh only showed no fluorescence. In the presence of both GSTP1-1 and GSH, fluorescence was dramatically increased and about 60% of DNs-Rh

was converted to rhodamine 110 at 30 min. However, in the absence of GSTP1, fluorescent changeby the free thiol of GSH was much slow (3% at 30 min).

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Fig.3 Monitoring intracellular GST activity in live cells
HeLa cells were treated with 2.5 μM of DNs-Rh for 30 min.In the case of addition of CNBSF, a potent irreversible GST inhibitor, cells were pre-treated with 1 mM CNBSF for 15 min prior to DNs-Rh addition. After DNs-Rh incubation, the cells were washed and observed fluorescent microscopy (Ex. 480nm/Em.535nm). CNBSF is also available from Funakoshi, catalog #FDV-0031(See below “Related product”). 

NOTE: Using this probe, you can observe the intracellular localization of rhodamine 110 converted from DNs-Rh by GSTs.
Fluorescent intensity is corresponding to cellular GST activity. However, localization is not equal to localization of GSTs.
 
Reference
  1. Shibata, A.,et al., Bioorg. Med. Chem. Lett., 18, 2246-2249 (2008) [PMID:18358719] "Rhodamine-based fluorogenic probe for imaging biological thiol."
  2. Alander, J., et al., Anal. Biochem., 390, 52-56 (2009) [PMID:19348782] "Characterization of a new fluorogenic substrate for microsomal glutathione transferase 1." 
  3. Zhang, J.,et al., J. Am. Chem. Soc., 133, 14109-14119 (2011) [PMID:21786801] "Synthesis and characterization of a series of highly fluorogenic substrates for glutathione transferases, a general stategy." 
  4. Shishido, Y., et al.,ChemBioChem., (in press) "A covalent inhibitor for Glutathione S-Transferase Pi (GSTP1-1) in human cells."
 
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