Description
Biotin Assay Kit (BA0211) (BA0211)
The Biotin Assay Kit (SKU: BA0211) provides a simple, colorimetric method for the quantitative determination of biotin. The assay is based on avidin's weak affinity for 4'-hydroxyazobenzene-2-carboxylic acid (HABA) and its strong affinity for biotin; the coloured avidin-HABA complex reagent has an absorbance at 500 nm, and when biotin binds to the avidin it displaces the HABA, causing a decrease in absorbance that is directly proportional to the biotin in the sample. Fast and sensitive, the kit offers a linear detection range of 8 to 200 uM biotin with a 30 uL sample in a 96-well plate. The procedure is convenient, involving addition of a single working reagent and immediate reading of the absorbance. As an add-mix-read type assay it is high-throughput and can be readily automated as a 96-well or 384-well plate assay for thousands of samples per day.
| Product Name: | Biotin Assay Kit (BA0211) |
| SKU: | BA0211 |
| Detection Method: | Colorimetric (OD 500 nm, 470-520 nm) |
| Detection Range: | 8 - 200 uM biotin (30 uL sample, 96-well) or 10 - 200 uM biotin (10 uL sample, 384-well) |
| Sample Type: | Foods, cosmetic products, supplements, biotinylated proteins and antibodies |
| Species Reactivity: | All |
| Assay Time: | 20 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Shipped at room temperature. Store all components at -20 degrees C upon receipt. |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
Biotin, or vitamin B7, is a water-soluble vitamin involved in metabolism, cell growth and protein synthesis. It is a cofactor to multiple carboxylases necessary for metabolising fatty acids, glucose and amino acids. Biotin is found in a wide range of foods and is often taken as a dietary supplement. In the biotechnology industry, biotin is commonly conjugated to proteins in a process called biotinylation; these biotinylated proteins can then be specifically selected using streptavidin or avidin's strong affinity for biotin in biochemical assays such as ELISAs. This kit is based on avidin's weak affinity for HABA and strong affinity for biotin. The avidin-HABA complex reagent has an absorbance at 500 nm; when the coloured reagent is introduced to biotin, the biotin binds to avidin, displacing the HABA and causing a decrease in absorbance that is directly proportional to the biotin in the sample.
- Fast and sensitive. Linear detection range: 8 to 200 uM biotin with 30 uL sample (96-well) or 10 to 200 uM biotin with 10 uL sample (384-well).
- Convenient. The procedure involves adding a single working reagent and reading the absorbance immediately.
- High-throughput. Add-mix-read type assay. Can be readily automated as a high-throughput 96-well or 384-well plate assay for thousands of samples per day.
- Direct assays of biotin in foods, cosmetic products and supplements.
- Direct assays of biotinylated proteins or antibodies.
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 | Prepare 500 uL of 200 uM Premix by mixing 10 uL of the 10 mM Standard and 490 uL of water, then dilute the standards in 1.5-mL centrifuge tubes as described in the standard table (200, 120, 60 and 0 uM). |
| 2 | Transfer 30 uL of each standard into separate wells of a clear, flat-bottom 96-well plate and 30 uL of each sample into separate wells. |
| 3 | Prepare sufficient Working Reagent (WR) by mixing, for each well, 300 uL of Reagent A and 15 uL of Reagent B. |
| 4 | Add 300 uL of Working Reagent to the four standards and the sample wells, tap the plate to mix briefly and thoroughly, and incubate for 20 minutes at room temperature. |
| 5 | Read the optical density at 500 nm (470-520 nm). |
| 6 | For the 384-well procedure, prepare standards as above, transfer 10 uL of standards and samples into wells, add 100 uL of Working Reagent (100 uL Reagent A and 5 uL Reagent B per well), incubate 20 minutes and read at 500 nm. |
Subtract the standard values from the blank value (#4) and plot the delta-OD against standard concentrations. Determine the slope and calculate the biotin concentration of a sample as: [Biotin] = [(ODBLANK - ODSAMPLE) / Slope (per uM)] x n (uM), where ODBLANK and ODSAMPLE are the optical density readings of the blank (#4) and sample respectively, and n is the dilution factor. If the calculated biotin concentration is higher than 200 uM, dilute the sample in water and repeat the assay. Conversions: 1 uM biotin equals 24.43 ug/dL or 244.3 ppb.
| Component | Quantity | Storage |
| Reagent A | 35 mL | -20 degrees C |
| Reagent B | 1.8 mL | -20 degrees C |
| Standard (10 mM Biotin) | 120 uL | -20 degrees C |
