Sialic Acid Assay Kit, BioAssay(TM), Colorimetric/Fluorimetric, High Sensitivity

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.\n\nSimple, direct and automation-ready procedures for measuring sialic acid concentrations find wide applications in research and drug discovery. Sialic acid assay uses a single Working Reagent that combines NANA aldolase, pyruvate oxidase and hydrogen peroxide determination in one step. The color intensity of the reaction product at 570nm or fluorescence intensity at lem/ex=585/530nm is directly proportional to sialic acid concentration in the sample.\n\nKey Features:\nSensitive and accurate. Use as little as 10ul samples. Linear detection range in 96-well plate: 0.02 to 1mM sialic acid for Colorimetric Assay:s and 2 to 100uM for Fluorimetric Assay:s. \nSimple and convenient. Can detect free sialic acid by addition of a single working reagent and incubation for 60 min at room temperature or total sialic acid by pre-treating samples with a 60 min hydrolysis step.\n\nApplications:\nDirect Assays: sialic acid in biological samples.\n\nKit Contents:\nAssay Buffer: 10ml Hydrolysis Reagent: 10ml\nEnzyme: 120ul Neutralization Reagent: 5ml\nDye Reagent: 120ul Standard: 500ul 10mM Sialic Acid\nStorage conditions. The kit is shipped on dry ice. Store all reagents at -20 degrees C. Shelf life of three months after receipt.\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nBound Sialic Acid Hydrolysis Procedure:\nNote: For measurement of free sialic acid, this procedure should be skipped.\n1. Combine 20ul of sample with 80ul Hydrolysis Reagent in a microcentrifuge tube (screw cap tube is preferable) and incubate at 80 degrees C for 60 min.\n2. Allow sample to cool to room temperature and briefly centrifuge at 14000 rpm to spin down the hydrolysis mixture.\n3. Add 20ul Neutralization Reagent to each hydrolysis reaction, briefly vortex to mix and briefly centrifuge at 14000 rpm to spin down the reaction. The samples are now ready for the sialic acid assay.\n\nColorimetric Procedure:\nNote: SH-group containing reagents (e.g. mercaptoethanol, DTT) may interfere with this assay and should be avoided in sample preparation.\n1. Equilibrate all components to room temperature. Prepare a 1mM Standard Premix by mixing 50ul of the 10mM Standard and 450ul dH2O. Dilute Standard in distilled water as follows.\nNo Premix+H2O Vol (ul) Sialic Acid (mM)\n1 100ul+0ul 100 1.0\n2 60ul+40ul 100 0.6\n3 30ul+70ul 100 0.3\n4 0ul+100ul 100 0\nTransfer 10ul standards and 10ul samples into separate wells of a clear flat-bottom 96-well plate.\n2. For each reaction well, mix 93ul Assay Buffer, 1ul Dye Reagent and 1ul Enzyme in a clean tube. Transfer 90ul Working Reagent into each assay well. Tap plate to mix. Freeze unused reagents for future use.\n3. Incubate 60 min at room temperature. Read optical density at 570nm (550-585nm). Note: if the Sample OD is higher than the Standard OD at 1mM, dilute sample in water and repeat the assay. Multiply result by the dilution factor.\n\nCalculation:\nSubtract blank OD (water, #4) from the standard OD values and plot the OD against standard concentrations. Determine the slope using linear regression fitting. The sialic acid concentration of a Sample is calculated as\n[Sialic Acid] =\nODSAMPLE

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SPECIFICATIONS

Catalog Number

S1013-31Q

Size

1Kit

References

1. Sugahara, K. et al. (1980). Enzymatic Assay of Serum Sialic Acid. Clinica Chimica Acta 108: 493-8.\n2. Simpson, H. et al. (1993). Serum sialic acid enzymatic assay based on microtitre plates: application for measuring capillary serum sialic acid concentrations. Br J Biomed Sci. 50: 164-7.