Description
Phospholipid Assay Kit (Colorimetric/Fluorometric) (BA0143) (BA0143)
The Phospholipid Assay Kit (SKU: BA0143) provides a simple, direct and high-throughput method for measuring choline-containing phospholipids in biological samples. Phospholipids are a major component of cell membranes and play important roles in signal transduction, with most containing one diglyceride, a phosphate group and one choline. In this assay phospholipids such as lecithin, lysolecithin and sphingomyelin are enzymatically hydrolysed 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 phospholipid concentration in the sample. The kit uses as little as 20 uL of sample and is suitable for both colorimetric and fluorometric detection.
| Product Name: | Phospholipid Assay Kit (Colorimetric/Fluorometric) (BA0143) |
| SKU: | BA0143 |
| Detection Method: | Colorimetric / Fluorometric |
| Detection Range: | Colorimetric 3 - 200 uM; fluorometric 0.6 - 20 uM phospholipid |
| Sample Type: | Serum, non-EDTA plasma and other 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 |
A convenient enzymatic assay for the quantitative colorimetric or fluorometric determination of choline-containing phospholipids. Phospholipids are hydrolysed to choline, which reacts with choline oxidase and a hydrogen-peroxide-specific dye to yield a coloured product measured at 570 nm or a fluorescent product measured at 530/585 nm. The method uses small sample volumes and is amenable to high-throughput screening.
- Sensitive, using as little as 20 uL of sample
- Linear detection range: colorimetric 3 - 200 uM, fluorometric 0.6 - 20 uM phospholipid
- Choice of colorimetric or fluorometric detection
- Suitable for high-throughput 96-well plate assays
- Determination of phospholipid in biological samples such as serum and non-EDTA plasma
- Studies of the effects of drugs on choline-containing phospholipid 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 standards: mix 24 uL 2 mM Standard with 216 uL water (200 uM) and dilute to 200, 120, 60 and 0 uM. Transfer 20 uL of each into wells of a clear flat-bottom 96-well plate (or a black plate for the fluorometric assay). |
| 4 | Transfer 20 uL of each sample into separate wells. If a sample is known to contain choline, prepare an additional Sample Blank well. |
| 5 | Prepare Working Reagent per well by mixing 85 uL Assay Buffer, 1 uL PLD Enzyme, 1 uL Enzyme Mix and 1 uL Dye Reagent. For Sample Blank wells, omit the PLD Enzyme. |
| 6 | Add 80 uL Working Reagent to each well, tap to mix and incubate for 30 min at room temperature. |
| 7 | Read optical density at 570 nm (550-585 nm). For the fluorometric assay use 0, 6, 12 and 20 uM standards and a black plate, reading fluorescence at 530/585 nm. |
Subtract the blank value from the standard values and plot the change in optical density or fluorescence against standard concentration. Determine the slope and calculate [Phospholipid] = ((R_SAMPLE - R_BLANK) / Slope) x n (uM), where R is the reading of the sample and the water or sample blank, and n is the dilution factor. If the result exceeds the linear range, dilute the sample in 0.5% Triton X-100 and repeat.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20 C |
| Enzyme Mix | Dried | -20 C |
| PLD Enzyme | 120 uL | -20 C |
| Dye Reagent | 120 uL | -20 C |
| Standard (2 mM phosphatidylcholine) | 400 uL | -20 C |
