Description
Choline Assay Kit (BA0088) (BA0088)
The Choline Assay Kit (SKU: BA0088) provides a simple, direct and high-throughput procedure for measuring choline in biological samples. Choline and its metabolites play important roles in membrane structure integrity, cellular signalling and cholinergic neurotransmission, and aberrant choline metabolism has been associated with mental illness such as anxiety. In this assay, free choline is oxidised by choline oxidase to betaine and hydrogen peroxide, which reacts with a specific dye to form a pink coloured product. The colour intensity at 570 nm, or fluorescence intensity at 530/585 nm, is directly proportional to the choline concentration in the sample.
| Product Name: | Choline Assay Kit (BA0088) |
| SKU: | BA0088 |
| Detection Method: | Colorimetric or Fluorometric |
| Detection Range: | Colorimetric 1 to 100 uM; fluorometric 0.2 to 10 uM choline |
| Sample Type: | Serum, plasma, urine, saliva, milk, tissue and cell culture |
| Species Reactivity: | All |
| Assay Time: | 30 minutes at room temperature |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Quantitative colorimetric or fluorometric determination of choline. Choline oxidase converts choline to betaine and hydrogen peroxide, which reacts with a dye to form a pink product read at 570 nm or by fluorescence at 530/585 nm.
- Uses 20 uL sample
- Colorimetric linear detection range 1 to 100 uM
- Fluorometric linear detection range 0.2 to 10 uM choline
- Simple room-temperature assay suitable for a wide range of samples
- Assays of choline in biological samples such as serum, plasma, urine, saliva, milk, tissue and cell culture
- Drug discovery and pharmacology: effects of drugs on choline 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 | Sample treatment. Assay serum and plasma directly. Prepare tissue and cell lysates by homogenisation in cold PBS and centrifugation (5 minutes at 14,000 rpm); use clear supernatants. For milk, mix 600 uL milk with 100 uL 6N HCl, centrifuge, transfer 300 uL supernatant to a clean tube and neutralise with 50 uL 6N NaOH (dilution factor n = 1.36). Keep SH-containing reagents below 5 uM. |
| 2 | Equilibrate all components to room temperature and briefly centrifuge tubes; keep thawed tubes on ice during the assay. Reconstitute the Enzyme Mix with 120 uL Assay Buffer (stable for 1 month at -20C); if a yellow precipitate forms after thawing, pellet it by centrifuging for 2 minutes at 14,000 rpm and use the clear supernatant. |
| 3 | Standards. Mix 12 uL 2 mM standard with 228 uL distilled water (final 100 uM) and dilute in distilled water as shown in the dilution table. Transfer 20 uL diluted standards into separate wells of a clear flat-bottom 96-well plate, and 20 uL of each sample into separate wells. |
| 4 | Colour reaction. Prepare working reagent by mixing, per reaction well, 85 uL Assay Buffer, 1 uL Enzyme Mix and 1 uL Dye Reagent. Add 80 uL working reagent to each well, tap to mix and incubate 30 minutes at room temperature. |
| 5 | Read optical density at 570 nm (550-585 nm). |
| 6 | Fluorometric assay. Use 0, 3, 6 and 10 uM choline standards in a black 96-well plate and read fluorescence at excitation 530 nm and emission 585 nm; the fluorometric assay is about ten times more sensitive than the colorimetric method. |
Subtract the blank value (#4) from the standard values and plot dOD or dF against standard concentrations to determine the slope. [Choline] = (R-sample - R-blank) / slope x n (uM), where R is the optical density or fluorescence reading and n is the sample dilution factor. Conversions: 1 mM choline equals 10.4 mg/dL, 0.010% or 104 ppm. If the calculated concentration exceeds 100 uM (colorimetric) or 10 uM (fluorometric), dilute the sample in water, repeat and multiply by the dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| Enzyme Mix | Dried | -20C |
| Dye Reagent | 120 uL | -20C |
| Standard (2 mM Choline) | 400 uL | -20C |
