Dehydrogenase-Glo™ Detection System
Sensitive Detection of Dehydrogenase Enzyme Activity
- Bioluminescent NAD(P)H detection toolkit that can be implemented rapidly to measure dehydrogenase activity in biological and biochemical samples
- Extremely sensitive detection from just 10s of cells
- Enables functional analysis of all major metabolic pathways
Catalog Number:
Size
Catalog Number: J9010
Catalog Number: J9020
Simple Method to Measure Endogenous Dehydrogenase Activity
The Dehydrogenase-Glo™ Detection System provides a core bioluminescent NAD(P)H detection technology that can be used to measure dehydrogenase activity in cell lysates, tissue homogenates and other samples. The Dehydrogenase-Glo™ Detection System provides only the reagents required for bioluminescent NAD(P)H detection. To build a specific dehydrogenase assay, you will need to identify and source the dehydrogenase substrate. We provide straightforward instructions for assay optimization in the technical manual.
How the Dehydrogenase-Glo™ Detection System Works
The core technology provided in the Dehydrogenase-Glo™ Detection System measures NAD(P)H. To measure a dehydrogenase using this system, you need to identify and source an appropriate substrate. The endogenous dehydrogenase couples substrate oxidation to NAD(P)+ reduction. The reduced dinucleotides, NAD(P)H, are measured using a luciferase reaction. The amount of light produced is proportional to the NAD(P)H produced from specific dehydrogenase activity in the sample.
Functional Analysis of Metabolic Pathways Using Dehydrogenase-Glo™ Detection System
The Dehydrogenase-Glo™ Detection System can be used with commercially available substrates to characterize metabolic pathway activity.
|
Pathway |
Dehydrogenase |
Dehydrogenase Substrate |
Dinucleotide (NAD/NADP) |
|
Glycolysis |
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) |
d-glyceraldehyde 3-phosphate (Sigma Cat.# 39705) |
NAD |
|
Pentose Phosphate Pathway |
Glucose 6-phosphate dehydrogenase (G6PDH) |
Glucose 6-phosphate, (Sigma Cat.# 10127645001) |
NADP |
|
Pentose Phosphate Pathway |
6-phosphogluconate dehydrogenase (6PGDH) |
6-phosphogluconate (Sigma Cat.# P7877) |
NADP |
|
TCA Cycle |
Isocitrate dehydrogenase (IDH) |
d-isocitrate, (Sigma Cat.# 58790) |
NADP |
Measuring Dehydrogenase Activity in K562 Cells
Measuring dehydrogenase activity in K562 cells. K562 cells were diluted twofold starting at 2.5 × 104 cells/ml in PBS, and 50μl was transferred into a 96-well assay plate. Dehydrogenase detection reagent was prepared for each dehydrogenase, and 50μl was added to sample wells. No-substrate controls (NAD only and NADP only) were included to determine background from nonspecific enzyme activity in K562 cells. Panel A. Luminescence was measured for dehydrogenases and controls after 60 minutes at different cell densities. Panel B. Time-dependent increase in GAPDH activity was shown at different cell densities. Luminescence was read every 15 minutes for 120 minutes. Solid black lines indicate linear range for each cell number. Panel C. Specific activity for each dehydrogenase was calculated. Data represent the average of three replicates with error bars indicating one standard deviation.
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Protocols
Specifications
Catalog Number:
What's in the box
| Item | Part # | Size | Concentration |
|---|---|---|---|
Reductase |
G884A | 1 × 55μl | 6mg/ml |
Reductase Substrate |
G885A | 1 × 55μl | |
Luciferin Detection Solution |
J135A | 1 × 5ml | |
NAD |
J136B | 1 × 275μl | |
NADP+ |
J154B | 1 × 100μl |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
U.S. Pat. Nos. 9,273,343 and 9,951,372, European Pat. No. 2751089, Japanese Pat. No. 6067019 and other patents pending.
What's in the box
| Item | Part # | Size | Concentration |
|---|---|---|---|
Reductase |
G884B | 1 × 275μl | 6mg/ml |
Reductase Substrate |
G885B | 1 × 275μl | |
Luciferin Detection Solution |
J135B | 1 × 50ml | |
NAD |
J136C | 1 × 1ml | |
NADP+ |
J154C | 1 × 500μl |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
U.S. Pat. Nos. 9,273,343 and 9,951,372, European Pat. No. 2751089, Japanese Pat. No. 6067019 and other patents pending.
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