Description
Sialic Acid Assay Kit (Colorimetric or Fluorometric) (BA0058) (BA0058)
The Sialic Acid Assay Kit (Colorimetric or Fluorometric) (SKU: BA0058) provides a sensitive method for the quantitative determination of free and total sialic acid in biological samples. Sialic acid is a general name for nine-carbon acidic sugars, the most common of which is N-acetylneuraminic acid (NANA), and it is widely distributed throughout mammalian tissues and fluids including serum. Sialylated oligosaccharides exhibit antiviral properties and influence blood coagulation and cholesterol levels, and the sialic acid level in body fluids is an important marker for diagnosing cancer. The assay uses an improved Warren method, in which sialic acid is oxidised to formylpyruvic acid, which reacts with thiobarbituric acid to form a pink-coloured product. The colour intensity at 549nm, or fluorescence intensity at excitation/emission 555/585nm, is directly proportional to the sialic acid concentration. As little as 60 µL of sample is required, and the fluorimetric assay is ten-fold more sensitive than the colorimetric assay.
| Product Name: | Sialic Acid Assay Kit (Colorimetric or Fluorometric) (BA0058) |
| SKU: | BA0058 |
| Detection Method: | Colorimetric or Fluorometric |
| Detection Range: | 5 to 1000 µM sialic acid (colorimetric); 0.5 to 100 µM (fluorimetric) in 96-well plate |
| Sample Type: | Serum, plasma, saliva, milk |
| Species Reactivity: | All |
| Assay Time: | Approximately 2.5 hours (60-minute hydrolysis, 60-minute oxidation, 10-minute colour reaction) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | The kit is shipped at ambient temperature. Store the Standard at -20°C and all other components at room temperature. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Room Temperature |
This kit uses an improved Warren method, in which sialic acid is oxidised to formylpyruvic acid, which reacts with thiobarbituric acid to form a pink-coloured product. The colour intensity at 549nm, or fluorescence intensity at λem/ex = 585/555nm, is directly proportional to the sialic acid concentration in the sample.
- Sensitive and accurate: use as little as 60 µL samples; linear detection range 5 to 1000 µM sialic acid for colorimetric assays and 0.5 to 100 µM for fluorimetric assays in 96-well plate
- Determines both free and total sialic acid
- The fluorimetric assay is ten-fold more sensitive than the colorimetric assay
- Direct assays: sialic acid in biological samples (e.g. serum, plasma, saliva, milk)
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 | Standards: equilibrate all components to room temperature. Prepare a 1000 µM Premix by mixing 25 µL of the 10 mM Standard and 225 µL distilled water, then dilute as shown in the table. Transfer 20 µL standards into labelled Eppendorf tubes and add 5 µL 10% TCA. |
| 2 | Sample treatment: for total sialic acid (TSA), mix 20 µL sample, 40 µL dH2O and 40 µL Hydrolysis Reagent, heat at 80°C for 60 minutes, cool, add 25 µL 10% TCA, vortex and centrifuge, then transfer 25 µL supernatant. For free sialic acid (FSA), mix 40 µL sample and 10 µL 10% TCA, centrifuge and transfer 25 µL supernatant. |
| 3 | Oxidation: prepare working reagent per tube (15 µL Hydrolysis Reagent, 50 µL dH2O and 65 µL Oxidation Reagent), add 125 µL to each tube and stand 60 minutes at room temperature. |
| 4 | Colour reaction: add 50 µL Dye Reagent to each tube, mix and heat 10 minutes at 100°C, cool 5-10 minutes, add 100 µL DMSO, mix and centrifuge 5 minutes at 14,000 rpm. Transfer 250 µL supernatant into wells of a clear flat-bottom 96-well plate. |
| 5 | Read optical density at 549nm (540-555nm). For the fluorimetric procedure, transfer to a black plate and read fluorescence at λex = 555nm and λem = 585nm. |
Subtract the blank value (#4) from the standard values and plot ∆OD or ∆F against standard concentrations. Determine the slope and calculate sialic acid concentration = [(RSAMPLE − RBLANK) / Slope] × n (µM), where R is the OD or fluorescence reading and n is the dilution factor (5 for TSA, 1 for FSA). Conversion: 1000 µM NANA equals 30.9 mg/dL or 309 ppm.
| Component | Quantity | Storage |
| Dye Reagent | 6 mL | Room temperature |
| Oxidation Reagent | 10 mL | Room temperature |
| 10% TCA | 5 mL | Room temperature |
| Hydrolysis Reagent | 10 mL | Room temperature |
| DMSO | 12 mL | Room temperature |
| Standard (10 mM Sialic Acid) | 500 µL | -20°C |
