Description
Bile Acid Assay Kit (BA0099) (BA0099)
The Bile Acid Assay Kit (SKU: BA0099) offers a convenient fluorometric method for the quantitative determination of total bile acids in biological samples. Bile acids are physiologically important molecules whose levels in serum, urine, faeces and bile can serve as markers for conditions such as hyperlipidaemia, cholestasis, gallstones and colon cancer. In this assay, 3-alpha-hydroxysteroid dehydrogenase reacts with the twelve non-sulphated bile acids, converting NAD to NADH, which reduces a probe to a highly fluorescent product. The resulting fluorescence intensity, measured at 530/585 nm, is proportional to the bile acid concentration in the sample. The kit does not measure bile acid-sulphates. This homogeneous mix-incubate-measure format is well suited to high-throughput screening.
| Product Name: | Bile Acid Assay Kit (BA0099) |
| SKU: | BA0099 |
| Detection Method: | Fluorometric |
| Detection Range: | 1 - 150 uM bile acids |
| Sample Type: | Serum, urine, faeces, bile and other biological samples |
| Species Reactivity: | All |
| Assay Time: | 20 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
This kit provides a homogeneous, non-radioactive fluorometric assay for total bile acids. An internal standard is used per sample to correct for variability in background and recovery between different sample matrices.
- Safe, non-radioactive assay
- Sensitive and accurate with a linear detection range of 1 - 150 uM bile acids
- Convenient, high-throughput homogeneous mix-incubate-measure format
- No wash or reagent transfer steps required
- Readily automated for high-throughput screening
- Determination of bile acids in serum, urine and other biological samples
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 | Use black flat-bottom plates. Bring all reagents to room temperature; briefly centrifuge enzyme tubes and keep on ice during the assay. Three wells are needed per sample: Sample, Internal Standard and Sample Blank. |
| 2 | Internal Standard: prepare 250 uL of 80 uM sodium cholate by mixing 20 uL of standard with 230 uL distilled water. |
| 3 | Transfer 20 uL of sample to each of the three wells. |
| 4 | Add 5 uL distilled water to the Sample and Sample Blank wells, and 5 uL Internal Standard to the Internal Standard well. |
| 5 | Working Reagent: for Internal Standard and Sample wells, mix per well 75 uL Assay Buffer, 8 uL NAD, 4 uL Probe, 1 uL Enzyme A and 1 uL Enzyme B. For Sample Blank wells, prepare Blank Reagent per well from 75 uL Assay Buffer, 8 uL NAD, 4 uL Probe and 1 uL Enzyme B (no Enzyme A). Add 80 uL of the appropriate reagent to each well. |
| 6 | Tap the plate to mix. Incubate for 20 minutes in the dark and read fluorescence intensity at 530/585 nm. |
[Bile Acids] = (F_SAMPLE - F_BLANK) / (F_STANDARD - F_SAMPLE) x 20 x n (uM), where the F values are the fluorescence intensities of the Sample, Internal Standard and Sample Blank wells, 20 uM is the effective internal standard concentration and n is the dilution factor. If the sample exceeds 150 uM, dilute in water, repeat and multiply by the dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| NAD Solution | 1 mL | -20C |
| Probe | 750 uL | -20C |
| Enzyme A | 120 uL | -20C |
| Enzyme B | 120 uL | -20C |
| Standard | 120 uL | -20C |
