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Fluoro NAD™ 
Fluorescent NAD/NADH Detection Kit - Detection of NAD/NADH in tissue extracts/cell samples
Key Benefits:
  • Detection of NAD/NADH content in cells or tissue extracts.
  • Detection of NAD/NADH levels in apoptosis, metabolism, proliferation, DNA repair, senescence, endocrine signaling and life span.
  • NAD/NADH detection in Bacterial, fungal and plant cells.
  • Fluorescent 96 well Plate Reader Readout (excitation: 530-570nm and emission at 590-600nm).
Introduction:

The role of pyridine nucleotides (NAD/NADH) metabolism in health, is of continual and increased interest. A growing amount of evidence supports the fact that NAD metabolism regulates important biological effect including life span. NAD, through poly-ADP-ribosyl polymerase (PARP), mono-ADP-ribosyltransferase (ARTs) and recently characterized sirtuin enzymes, exerts potential biological effects. These enzymes modify proteins to regulate their function via ADP-ribosylation or deacetylation and are involved in several pathways including apoptosis, DNA repair, senescence and endocrine signaling. This suggests that either the enzymes or NAD could be an important therapeutical target 1.
Assay Principle:

The Fluoro NAD/NADH detection kit utilizes a non-fluorescent detection reagent, which is reduced in the presence NADH to produce its fluorescent analog and NAD. NAD is further converted to NADH via an enzyme coupled reaction. The enzyme reaction specifically reacts with NAD/NADH and not with NADP/NADPH

Reaction:

1. NADH + non-fluorescent detection reagent+ electron coupler -----> fluorescent 

analog + NAD

2. NAD + enzyme coupled reaction -------> NADH (then proceeds to reaction 1).

excitation: 530-570nm and emission at 590-600nm
FluoroNADNADH


Figure 1. NADH standard curve titrated in NAD/NADH lysis buffer. Incubation time = 1 hour.

FluoroNADNADH


Figure 2. NAD/NADH Assay showing no cross-reactivity with NADPH. Incubation time = 1 hour.

FluoroNADNADH


NAD and NADH levels were assayed using the Fluoro NAD/NADH kit. Jurkat cells 6 X105 and K562 cells 7 X105 per tube (n=3).

 

Kit contents (for 100 assays)
1. Enzyme Mix .................................................. Part# 6021 - Store at -20C
2. NADH Detection Reagent ............................. Part# 4018 - Store at -20C
3. NADH Standard ............................................. Part# 7013 - Store at -20C
4. NAD/NADH Reaction Buffer ........................ Part# 3044 - Store at +2-8 C
5. NAD/NADH Lysis Buffer .............................. Part# 3045 - Store at +2-8 C
6. NAD Extraction Buffer .................................. Part# 3046 - Store at +2-8 C
7. NADH Extraction Buffer ................................ Part# 3047 - Store at +2-8 C
8. NADH Standard Diluent ................................ Part# 3051 - Store at +2-8 C

Product Catalog # Size*
Fluoroscent NAD/NADH Detection Kit FLNADH 100-2 100
Fluoroscent NAD/NADH Detection Kit FLNADH 100-3 500