Description
Free Fatty Acid Measurement Kit (BA0101) (BA0101)
The Free Fatty Acid Measurement Kit (SKU: BA0101) provides a simple, single-step and high-throughput method for quantifying free fatty acids in biological and food samples. Fatty acids play important roles in cellular synthesis and energy metabolism and are implicated in disorders such as diabetes mellitus. In the assay, free fatty acids are enzymatically converted to acyl-CoA and subsequently to hydrogen peroxide, which reacts with a specific dye to form a pink coloured product. The optical density at 570 nm, or the fluorescence intensity at 530/585 nm, is directly proportional to the free fatty acid concentration in the sample. The room-temperature mix-and-read procedure is readily automated.
| Product Name: | Free Fatty Acid Measurement Kit (BA0101) |
| SKU: | BA0101 |
| Detection Method: | Colorimetric (570 nm) / Fluorometric (530/585 nm) |
| Detection Range: | Colorimetric 7 - 1000 uM; fluorometric 7 - 100 uM fatty acid |
| Sample Type: | Serum, plasma, urine, saliva, milk, cell cultures, food and agricultural products |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A convenient colorimetric or fluorometric assay for free fatty acids using only 10 uL of sample. Enzyme A is supplied dried and is reconstituted before use.
- Sensitive, using only 10 uL of sample
- Colorimetric linear range 7 - 1000 uM and fluorometric range 7 - 100 uM
- Convenient room-temperature mix-and-read procedure
- Readily automated for high-throughput assays
- Assay of free fatty acids in serum, plasma, urine, saliva, milk, cell cultures, food and agricultural products
- Studying the effects of drugs on free fatty acid 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 | Reconstitute Enzyme A with 120 uL distilled water, dissolve fully and incubate at room temperature for 15 minutes. Store reconstituted Enzyme A at -20C and use within 2 months. |
| 2 | Colorimetric Assay: dilute blood samples 2- to 100-fold in water as appropriate. Equilibrate components to room temperature and keep thawed tubes on ice. The thawed Standard should be clear and colourless. |
| 3 | Standards: dilute the 1000 uM standard in Assay Buffer per the table. Transfer 10 uL of each diluted standard into wells of a clear flat-bottom 96-well plate. Add 10 uL of each sample to separate wells. |
| 4 | Colour reaction: prepare Working Reagent per well from 90 uL Assay Buffer, 1 uL Enzyme A, 1 uL Enzyme B, 1 uL CoSubstrate and 1 uL Dye Reagent. Add 90 uL to each well, tap to mix and incubate 30 minutes at room temperature. |
| 5 | Read optical density at 570 nm (550-585 nm). For the fluorometric assay, use 0, 30, 60 and 100 uM standards and a black plate, and read fluorescence at 530/585 nm. |
Subtract the blank value from the standard values and plot delta-OD or delta-F against standard concentration. Determine the slope and calculate [Free Fatty Acid] = (R_SAMPLE - R_BLANK) / Slope x n (uM), where R is the optical density or fluorescence reading and n is the sample dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 20 mL | -20C |
| Enzyme A | Dried | -20C |
| CoSubstrate | 120 uL | -20C |
| Dye Reagent | 120 uL | -20C |
| Enzyme B | 120 uL | -20C |
| Standard (1 mM palmitic acid) | 1 mL | -20C |
