Description
NAD/NADH Assay Kit (BA0106) (BA0106)
The NAD/NADH Assay Kit (SKU: BA0106) provides a sensitive fluorometric method for measuring NAD, NADH and their ratio in cell and tissue extracts. Pyridine nucleotides play a central role in metabolism and cellular redox state. The assay is based on a lactate dehydrogenase cycling reaction, in which the NADH formed reduces a probe to a highly fluorescent product; the fluorescence at 530/585 nm is proportional to the NAD+/NADH concentration. The assay is highly specific, with less than 1% interference from NADP+/NADPH, and is readily automated for high-throughput applications.
| Product Name: | NAD/NADH Assay Kit (BA0106) |
| SKU: | BA0106 |
| Detection Method: | Fluorometric (530/585 nm) |
| Detection Range: | 0.02 - 1 uM NAD+/NADH (detection limit 0.02 uM) |
| Sample Type: | Cell or tissue extracts |
| Species Reactivity: | All |
| Assay Time: | 10 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A highly specific fluorometric cycling assay for measuring NAD, NADH and their ratio, using separate extractions for the oxidised and reduced forms.
- Sensitive and accurate with a detection limit of 0.02 uM and linearity up to 1 uM
- Convenient single working reagent read at time zero and 10 minutes
- High-throughput and readily automated
- Highly specific with less than 1% interference from NADP+/NADPH
- Direct assay of NAD+/NADH concentrations and ratios in cell or tissue extracts
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Sample Preparation: for tissue weigh ~20 mg and wash in cold PBS; for cells wash and pellet ~10^5 cells. Homogenise in 100 uL NAD extraction buffer (for NAD) or NADH extraction buffer (for NADH). Heat at 60C for 5 min, add 20 uL Assay Buffer and 100 uL of the opposite extraction buffer to neutralise, vortex and centrifuge at 14,000 g for 5 min. Determining both NAD and NADH requires two separate extractions. |
| 2 | Calibration Curve: prepare 5000 uL of 1 uM NAD Premix by mixing 5 uL of 1 mM Standard with 4995 uL distilled water, then dilute per the table. Transfer 50 uL of each standard into a black flat-bottom 96-well plate. |
| 3 | Samples: add 50 uL of each sample to separate wells. |
| 4 | Reagent Preparation: for each well mix 40 uL Assay Buffer, 1 uL Enzyme A, 1 uL Enzyme B, 10 uL Lactate and 5 uL Probe. |
| 5 | Reaction: add 50 uL Working Reagent per well quickly and tap to mix. |
| 6 | Read fluorescence at 530/585 nm at time zero (F0) and after a 10-minute incubation at room temperature (F10), protected from light. |
Compute delta-F for each standard and sample by subtracting F0 from F10. Plot the standard delta-F values and determine the slope. [NAD(H)] = (delta-F_SAMPLE - delta-F_BLANK) / Slope x n (uM), where the blank is standard 4 and n is the dilution factor. If a sample delta-F exceeds that of the 1 uM standard, dilute in distilled water, repeat and multiply by the dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| Lactate | 1.5 mL | -20C |
| Probe | 750 uL | -20C |
| Enzyme A | 120 uL | -20C |
| Enzyme B | 120 uL | -20C |
| NAD Standard | 0.5 mL | -20C |
| NAD Extraction Buffer | 12 mL | -20C |
| NADH Extraction Buffer | 12 mL | -20C |
