ab83375 Sialic Acid (NANA) Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of Sialic Acid (NANA) in various samples. Version 2 Last Updated 4 March 2015
This product is for research use only and is not intended for diagnostic use. 1
Table of Contents 1. Overview 3 2. Protocol Summary 4 3. Components and Storage 5 4. Assay Protocol 7 5. Data Analysis 9 6. Troubleshooting 11 2
1. Overview 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. Abcam's Sialic Acid (NANA) 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 OxiRed probe to give fluorescence (Ex/Em=535/587 nm) and absorbance (OD = 570 nm). The kit measures sialic acid in the linear range of 0.1-10 nmol with a detection sensitivity ~1 µm concentration. 3
2. Protocol Summary Sample Preparation Standard Curve Preparation Prepare and Add Reaction Mix Measure Optical Density or Fluorescence 4
3. Components and Storage A. Kit Components Item Quantity Sialic Acid Assay Buffer 25 ml Sialic Acid Probe in DMSO 0.2 ml Sialic Acid Converting Enzyme (Lyophilized) 1 vial Sialic Acid Development Mix (Lyophilized) 1 vial Sialic Acid Standard (10 µmol, Lyophilized) 1 vial * Store the kit at -20 C, protect from light. Warm the Assay Buffer to room temperature before use. Briefly centrifuge vials prior to opening. Read the entire protocol before the assay. 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, DEVELOPING MIX: Reconstitute with 220 μl Sialic Acid Assay Buffer separately. Pipette 5
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 μl dh 2 O to generate 100 mm (100 nmol/μl) 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. B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker 6
4. Assay Protocol 1. 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 μl/well with Assay Buffer. We suggest testing several doses of your sample to make sure the readings are within the standard curve range. 2. Standard Curve Preparation: a. For the colorimetric assay: Dilute 10 μl of the 100 mm Sialic Acid Standard with 990 μl dh 2 O to generate 1 mm standard Sialic Acid. Add 0, 2, 4, 6, 8, 10 μl 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 μl with Assay Buffer. b. For the fluorometric assay: Dilute the standard to 0.1 mm (0.1 nmol/μl), then follow the same protocol as colorimetric assay. Will give 0, 0.2, 0.4, 0.6, 0.8, 1 nmol/well Standard. 7
3. Sialic Acid Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 μl Reaction Mix containing: Sialic Acid Measurement Background Control* Assay Buffer 44 μl 46 μl Sialic Acid Converting Enzyme 2 μl --- Sialic Acid Development Mix 2 μl 2 μl Sialic Acid Probe** 2 μl 2 μl * Note: Pyruvate will generate background. If a significant amount of pyruvate is suspected in your sample, you may do a background control. Do a 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. ** Note: For the fluorescent assay, dilute the probe 10X to reduce background reading. Add 50 μl of the Reaction Mix to each well containing the Triglyceride Standard, samples and controls. Mix well. Incubate at room temperature for 30 minutes, protect from light. 4. Add 50 μl of the Reaction Mix to each well containing the Sialic acid standard and test samples. Mix well. Incubate the reaction for 30 min at room temperature, protect from light 8
5. Measure OD at 570 nm or fluorescence at Ex/Em 535/587 nm in a microplate reader. 5. Data Analysis Correct background by subtracting the value derived from the zero 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: Concentration = Sa / Sv (nmol/μl or mm) Where: Sa is the Sialic acid content of unknown samples (in nmol) from standard curve. Sv is sample volume (in μl) added into the assay wells. Sialic acid Molecular Weight is 309.3 g/mol. 9
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6. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 11
Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) 12
Problem Reason Solution Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit For further technical questions please do not hesitate to contact us by email (technical@abcam.com) or phone (select contact us on www.abcam.com for the phone number for your region). 13
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UK, EU and ROW Email: technical@abcam.com Tel: +44 (0)1223 696000 www.abcam.com US, Canada and Latin America Email: us.technical@abcam.com Tel: 888-77-ABCAM (22226) www.abcam.com China and Asia Pacific Email: hk.technical@abcam.com Tel: 108008523689 ( 中國聯通 ) www.abcam.cn Japan Email: technical@abcam.co.jp Tel: +81-(0)3-6231-0940 www.abcam.co.jp Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. 15 All information / detail is correct at time of going to print.