data sheet - BioVision
Contents
1. Storage and Handling Store the kit at 20 C protect from light Warm Assay Buffer to room temperature before use Briefly centrifuge vials prior to opening Read the entire protocol before the assay Reagent Reconstitution and General Consideration Sialic Acid Probe Ready to use as supplied Warm to room temperature to melt frozen DMSO prior to use Protect from light and moisture Stable for 2 months at 20 C Sialic Acid Converting Enzyme Development Mix Reconstitute with 220 ul Sialic acid Assay Buffer separately Pipette up and down to dissolve Keep the Enzyme and Development Mix on ice during use Aliquot and store at 20 C if they will not all be used at once Avoid repeated freeze thaw cycles Use within two months Sialic Acid Standard Dissolve in 100 ul dH2O to generate 100 mM 100 nmol ul Sialic acid Standard Solution Keep on ice while in use Store at 20 C Ensure that the Assay Buffer is warmed to room temperature before the reaction Sialic Acid Assay Protocol Standard Curve For the Colorimetric Assay Dilute 10 ul of the 100 mM Sialic Acid Standard with 990 ul dH20 to generate 1 mM standard Sialic Acid Add 0 2 4 6 8 10 ul of the diluted Sialic Acid Standard into a 96 well plate to generate 0 2 4 6 8 10 nmol well standard Bring the volume to 50 ul with Assay Buffer For the Fluorometric Assay Dilute the standard to 0 1 mM 0 1 nmol ul then follow the same protocol as colorimetric assay Wi2. BioVision Sialic Acid NANA Colorimetric Fluorometric Assay Kit Catalog K566 100 100 Reactions Store kit at 20 C Introduction Sialic acid is a generic term for the N or O substituted derivatives of neuraminic acid a monosaccharide with a nine carbon backbone It is also the name for the most common member of this group N acetylneuraminic acid Sialic acids are found widely distributed in animal tissues and to a lesser extent in other species ranging from plants and fungi to yeasts and bacteria mostly in glycoproteins and gangliosides It has been shown recently that sialic acid level may be associated with developmental and pathological stages BioVision s Sialic Acid Assay Kit provides a simple and convenient means of measuring free Sialic Acid in a variety of biological samples The kit utilizes an enzyme coupled reaction in which free sialic acid is oxidized resulting in development of the Oxi Red probe to give fluorescence Ex Em 535 587 nm and absorbance OD 570 nm The kit measures sialic acid in the linear range of 0 1 to 10 nmol with a detection sensitivity 1 uM concentration Kit Contents Components K566 100 Cap Code Part No Sialic Acid Assay Buffer 25 ml WM K566 100 1 Sialic Acid Probe in DMSO 0 2 ml Red K566 100 2A Sialic Acid Converting Enzyme lyophilized Purple K566 100 4 Sialic Acid Development Mix lyophilized Green K566 100 5 Sialic Acid Standard 10 umol lyophilized Yellow K566 100 6
3. and freeze samples if needed to use multiple times Troubleshoot if needed deproteinize samples e Use fresh samples or store at correct temperatures till use Lower Higher readings in Samples and Standards e Improperly thawed components e Use of expired kit or improperly stored reagents e Allowing the reagents to sit for extended times on ice e Incorrect incubation times or temperatures e Incorrect volumes used e Thaw all components completely and mix gently before use e Always check the expiry date and store the components appropriately e Always thaw and prepare fresh reaction mix before use e Refer datasheet amp verify correct incubation times and temperatures e Use calibrated pipettes and aliquot correctly Readings do not follow a linear pattern for Standard curve e Use of partially thawed components e Pipetting errors in the standard e Pipetting errors in the reaction mix Air bubbles formed in well e Standard stock is at an incorrect concentration e Calculation errors e Substituting reagents from older kits lots e Thaw and resuspend all components before preparing the reaction mix e Avoid pipetting small volumes e Prepare a master reaction mix whenever possible Pipette gently against the wall of the tubes e Always refer the dilutions in the data sheet e Recheck calculations after referring the data sheet e Use fresh components from the same kit Unanticipated results e Measured at in
4. correct wavelength Samples contain interfering substances e Use of incompatible sample type Sample readings above below the linear range e Check the equipment and the filter setting Troubleshoot if it interferes with the kit e Refer data sheet to check if sample is compatible with the kit or optimization is needed e Concentrate Dilute sample so as to be in the linear range Note The most probable list of causes is under each problem section Causes Solutions may overlap with other problems BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 2 of 2
5. it Free Glycerol Assay Kit Hemin Assay Kit Glucose Assay Kit L Amino Acid Assay Kit Cholesterol Assay Kit HDL LDL Assay Kit Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 2 BioVision GENERAL TROUBLESHOOTING GUIDE rev 01 14 For research use only Problems Cause Solution Assay not working e Use of ice cold assay buffer e Omission of a step in the protocol e Plate read at incorrect wavelength e Use of a different 96 well plate e Assay buffer must be at room temperature e Refer and follow the data sheet precisely Check the wavelength in the data sheet and the filter settings of the instrument Fluorescence Black plates Luminescence White plates Colorimeters Clear plates Samples with erratic readings e Use of an incompatible sample type Samples prepared in a different buffer Samples were not deproteinized if indicated in datasheet Cell tissue samples were not completely homogenized e Samples used after multiple free thaw cycles e Presence of interfering substance in the sample e Use of old or inappropriately stored samples e Refer data sheet for details about incompatible samples e Use the assay buffer provided in the kit or refer data sheet for instructions e Use the 10 kDa spin cut off filter or PCA precipitation as indicated e Use Dounce homogenizer increase the number of strokes observe for lysis under microscope Aliquot
6. ll give 0 0 2 0 4 0 6 0 8 1 nmol well Standard Sample Preparation Samples can be tested for free sialic acid or hydrolyzed to measure bound sialic acid as well There are a variety of hydrolysis protocols and users should be cautious in selecting which protocol to use Once the sample has been prepared add samples to a 96 well plate and bring the volume to 50 ul well with Assay Buffer We suggest testing several doses of your sample to make sure the readings are within the standard curve range FOR RESEARCH USE ONLY Not to be used on humans BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 01 14 For research use only Mix enough reagents for the number of assays to be performed For each well prepare a total 50 ul Reaction Mix containing Sialic Acid Measurement 3 Reaction Mix Background Control Assay Buffer 44 ul 46 ul Sialic Acid Converting Enzyme 2u o Sialic Acid Development Mix 2 yl 2 ul Sialic Acid Probe 2 ul 2 ul Pyruvate will generate background If a significant amount of pyruvate is suspected in your sample you may do a background control Do pyruvate background control without Sialic Acid Converting Enzyme which will detect only endogenous pyruvate but not Sialic Acid The pyruvate background should be subtracted from Sialic Acid For the fluorescent assay dilute the probe 10X to reduce background reading Add 50 ul of the Reaction Mix to each well containi
7. ng the Sialic acid standard and test samples Mix well Incubate the reaction for 30 min at room temperature protect from light Measure OD at 570 nm or fluorescence at Ex Em 535 587 nm in a microplate reader Calculation Correct background by subtracting the value derived from the 0 Sialic Acid control from all sample and standard readings The background reading can be significant and must be subtracted from sample readings Plot Sialic Acid standard curve Apply sample readings to the standard curve Sialic Acid concentrations of the test samples can then be calculated C S S nmol l or mM Where Sais the Sialic acid content of unknown samples in nmol from standard curve S is sample volume in ul added into the assay wells Sialic acid Molecular Weight is 309 3 g mol 10000 y 0 1393x 0 003 3000 y 8912 7x 78 371 gt 6000 En a 4000 OD 570 nm 2000 0 0 2 4 6 8 10 0 0 2 0 4 0 6 0 8 1 nmol Sialic Acid nmol Sialic Acid RELATED PRODUCTS NAD P NAD P H Quantification Kit Ascorbic Acid Quantification Kit Total Antioxidant Capacity TAC Assay Kit Ethanol Assay Kit Pyruvate Assay Kit Creatinine Assay Kit Ammonia Assay Kit Triglyceride Assay Kit Choline Acetylcholine Quantification Kit Sarcosine Assay Kit Glycogen Assay Kit Phosphatase Assay Kits ADP ATP Ratio Assay Kit Glutathione Detection Kit Fatty Acid Assay Kit Uric Acid Assay Kit Lactate Assay Kit amp Il Nitric Oxide Assay K
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