Sialic Acid Assay Kit

Catalog No. ABIN1000277

Product Details

Target: Sialic Acid (SA)

Application: Biochemical Assay (BCA)

Sample Type: Serum, Plasma, Saliva, Milk

Characteristics: Sensitive and accurate. Use as little as 60 µL samples. Linear detection range in 96-well plate: 5 to 1000 µM sialic acid for colorimetric assays and 0.5 to 100 µM for fluorimetric assays.

Components: Dye Reagent: 6 mL. Oxidation Reagent: 10 mL. 10% TCA: 5 mL. Hydrolysis Reagent: 10 mL. DMSO: 12 mL. Standard: 500 µL 10 mM Sialic Acid.

Material not included: Pipeting devices, centrifuge tubes, centrifuge, heat block, clear flat- bottom 96-well plates, black 96-well plates (e.g. Corning Costar) and plate readers.

Application Details

Application Notes: Direct Assays: sialic acid in biological samples (e.g. serum, plasma, saliva, milk).

Protocol: 1. Standards. Equilibrate all components to room temperature. Prepare a 1000 µMsialic acid standard Premix by mixing 25 µL of the 10 mM Standard and 225 µL distilled water dH2O. No Premix + dH2O Vol (µL) Sialic Acid (µM) 1 100µL + 0µL 100 1000 2 60µL + 40µL 100 600 3 30µL + 70µL 100 300 4 0µL + 100µL 100 0 Transfer 20 µL standards into four labeled Eppendorf tubes, add 5 µL 10% TCA.
2. Samples treatment. To determine total sialic acid (TSA), samples need to be hydrolyzed to release bound sialic acid. In an Eppendorf tube, mix 20 µL sample, 40 µL dH2O and 40 µL Hydrolysis Reagent. Heat at 80°C for 60 min, let cool and briefly centrifuge. Add 25 µL 10% TCA, vortex and centrifuge at 14,000 rpm for 10 min. Transfer 25 µL supernatant into a clean tube and label it TSA. To determine free sialic acid (FSA), directly precipitate protein by mixing 40 µL sample and 10 µL 10% TCA. Vortex and centrifuge at 14,000 rpm for 10 min. Transfer 25 µL supernatant into a clean tube and label it FSA.
3. Oxidation. Prepare working reagent for each tube by mixing 15 µL Hydrolysis Reagent, 50 µL dH2O and 65 µL Oxidation Reagent. Add 125 µL working reagent to each tube and let stand for 60 min at room temperature.
4. Color Reaction. Add 50 µL Dye Reagent to each tube. Mix and heat for 10 min at 100°C. Let cool for another 5-10 min. Add 100 µL DMSO to each tube. Mix and centrifuge for 5 min at 14,000 rpm. Transfer 250 µL supernatant into separate wells of a clear, flat-bottom 96-well plate.
5. Read optical density at 549 nm (540-555nm).

Calculation of Results:

Subtract blank value (#4) from the standard values and plot the OD or F against standard concentrations. Note: if the Sample OD value is higher than that for the 1000 µM Standard, or sample fluorescence intensity higher than that for the 100 µM Standard, dilute sample in water and repeat the assay. Multiply result by the fold of dilution.
Conversions: 1000 µM NANA equals 30.9 mg/dL or 309 ppm.

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

Pawluczyk, Ghaderi Najafabadi, Patel, Desai, Vashi, Saleem, Topham: "Sialic acid attenuates puromycin aminonucleoside-induced desialylation and oxidative stress in human podocytes." in: Experimental cell research, Vol. 320, Issue 2, pp. 258-68, (2013) (PubMed).

Carvalho, de Oliveira, Freitas, Gonçalves, Santos: "Variations on fibrinogen-erythrocyte interactions during cell aging." in: PLoS ONE, Vol. 6, Issue 3, pp. e18167, (2011) (PubMed).

Target Details

Target: Sialic Acid (SA)

Alternative Name: Sialic Acid (SA Products)

Background: Quantitative determination of free and total sialic acid (NANA) by colorimetric (549nm) or fluorimetric (555nm/585nm) methods.
Procedure: 2.5 hrs.

Sialic Acid is a general name for nine carbon acidic sugars with N- or O-substituted derivatives. The most common member of these sugars is N-acetylneuraminic acid (NANA). Sialic acid is widely distributed throughout mammalian tissues and fluids including serum. Sialylated oligosaccharides have been shown to exhibit antiviral properties and are also known to influence blood coagulation and cholesterol levels. The sialic acid level in body fluids is also an important marker for diagnosing cancer. Simple and direct procedures for measuring sialic acid concentrations find wide applications in research and drug discovery. This sialic acid assay uses an improved Warren method, in which sialic acid is oxidized to formylpyruvic acid which reacts with thiobarbituric acid to form a pink colored product. The color intensity at 549 nm or fluorescence intensity at gamma em/ex = 585/555 nm is directly proportional to sialic acid concentration in the sample.