Description
Phospholipase D Activity Assay Kit (BA0144) (BA0144)
The Phospholipase D Activity Assay Kit (SKU: BA0144) provides a simple, high-throughput method for measuring phospholipase D (PLD) activity in biological samples. Phospholipase D catalyses the hydrolysis of the phosphodiester bond of glycerophospholipids to generate phosphatidic acid and a free head group, and abnormalities in its expression have been associated with human cancers. In this assay PLD hydrolyses phosphatidylcholine to choline, which is then determined using choline oxidase and a hydrogen-peroxide-specific dye. The optical density of the pink product at 570 nm, or the fluorescence intensity at 530/585 nm, is directly proportional to the PLD activity in the sample. The kit uses as little as 10 uL of sample and adds a single working reagent, making it well suited to drug-screening applications.
| Product Name: | Phospholipase D Activity Assay Kit (BA0144) |
| SKU: | BA0144 |
| Detection Method: | Colorimetric / Fluorometric |
| Detection Range: | Colorimetric 0.06 - 10 U/L; fluorometric 0.04 - 1 U/L |
| Sample Type: | Biological samples |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at -20 C. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
An enzyme-coupled assay for the quantitative colorimetric or fluorometric determination of phospholipase D activity. PLD hydrolyses phosphatidylcholine to choline, which reacts with choline oxidase and a hydrogen-peroxide-specific dye to yield a coloured or fluorescent product. The single-working-reagent format is readily adapted to high-throughput drug-screening assays.
- Sensitive, using as little as 10 uL of sample
- Detection range: colorimetric 0.06 - 10 U/L, fluorometric 0.04 - 1 U/L
- Simple single-working-reagent addition
- Readily adapted to high-throughput drug screening
- Direct measurement of phospholipase D in biological samples
- Studies of the effects of drugs on phospholipase D metabolism
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 | Equilibrate all components to room temperature and briefly centrifuge the tubes. Keep thawed tubes on ice during the assay. |
| 2 | Reconstitute Enzyme Mix with 120 uL Assay Buffer (stable one month at -20 C). If a yellow precipitate forms, centrifuge for 2 min at 14,000 rpm and use the clear supernatant. |
| 3 | Prepare calibrators: mix 33 uL Calibrator with 187 uL water (300 uM choline) and dilute to 300, 180, 90 and 0 uM. Transfer 10 uL of each into wells of a clear flat-bottom 96-well plate (or a black plate for the fluorometric assay). |
| 4 | Transfer 10 uL of each sample into separate wells. |
| 5 | Prepare Working Reagent per well by mixing 85 uL Assay Buffer, 1 uL Enzyme Mix, 1 uL Dye Reagent and 12 uL Substrate. Add 90 uL to each well and tap to mix. |
| 6 | Incubate at the desired temperature protected from light and read optical density at 570 nm (550-585 nm) at 10 and 30 min. For the fluorometric assay use 0, 9, 18 and 30 uM calibrators and read fluorescence at 530/585 nm. |
Subtract the blank from the standard values and plot the change in optical density or fluorescence against calibrator concentration. Determine the slope and calculate [Phospholipase D] = ((R30 - R10) / (Slope x 20)) x n (U/L), where R30 and R10 are the sample readings at 30 and 10 min, 20 is the reaction time in minutes and n is the dilution factor. One unit of PLD catalyses formation of 1 umole of choline per minute at pH 7.4.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20 C |
| Enzyme Mix | Dried | -20 C |
| Calibrator | 400 uL | -20 C |
| Dye Reagent | 120 uL | -20 C |
| Substrate | 1.5 mL | -20 C |
