For the rapid, sensitive and accurate detection of long-chain Free Fatty Acids in various samples.
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1 ab65341 Free Fatty Acid Quantification Kit Instructions for Use For the rapid, sensitive and accurate detection of long-chain Free Fatty Acids in various samples. This product is for research use only and is not intended for diagnostic use.
2 1
3 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
4 1. Overview Fatty Acids play very important roles in normal metabolism and many disease developments. They are precursors to a number of bioactive classes of compounds such as prostaglandins, leucotrienes and others, and have been implicated in diverse functions such as autism, immune system and inflammation response. Abcam s Free Fatty Acid Quantification Kit provides a convenient, sensitive enzyme-based method for detecting the long-chain free fatty acids in various biological samples, such as serum, plasma and other body fluids, food, growth media, etc. In the assay, Fatty Acids are converted to their CoA derivatives, which are subsequently oxidized with the concomitant generation of color or fluorescence. C-8 (octanoate) and longer fatty acids can then be easily quantified by either colorimetric (spectrophotometry at λ = 570 nm) or fluorometric (at Ex/Em = 535/587 nm) methods with detection limit 2 µm free fatty acid in variety samples. 3
5 2. Protocol Summary Sample Preparation Standard Curve Preparation Acyl-CoA Synthesis A Add Reaction Mix Measure Optical Density or Fluorescence 4
6 3. Components and Storage A. Kit Components Item Quantity Fatty Acid Assay Buffer 25 ml Fatty Acid Probe (in DMSO) 0.2 ml ACS Reagent 1 vial Enzyme Mix 1 vial Enhancer 200 µl Palmitic Acid Standard (1nmol/µl) 300 µl * Store kit at -20 C, protect from light. Allow Fatty Acid Assay Buffer warm to room temperature before use. FATTY ACID PROBE: Ready to use as supplied. Allow Probe to warm to room temperature prior to use to melt frozen DMSO. Store at -20 C, protect from light and moisture. Use within two months. ENZYMES: Dissolve ACS (Acyl-CoA Synthetase) Reagent and Enzyme Mix individually with 220 µl Assay Buffer each by pipetting up and down. Store at -20 C. Use within two months. 5
7 B. Additional Materials Required Microcentrifuge Micro-homogenizer Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker 6
8 4. Assay Protocol 1. Sample Preparation: a. For liquid samples: Different volumes of samples can be directly added to each well in a 96-well plate, then bring up the volume to 50 µl/well with Assay Buffer. For unknown samples, we suggest using different doses to ensure the readings are within the standard curve range. b. For cell or tissue samples: 10 6 cells or 10 mg tissue samples can be extracted by homogenization with 200 µl of chloroform-triton X-100 (1% Triton X-100 in pure chloroform) in a micro-homogenizer. Spin the extract 5-10 minutes at top speed in a microcentrifuge. Collect organic phase (lower phase), air dry at 50 C to remove chloroform. Vacuum dry 30 min to remove trace chloroform. Dissolve the dried lipids (in Triton X-100) in 200 µl of Fatty Acid Assay Buffer by vortexing extensively for 5 mins Note: The solution may be slightly turbid or opalescent, but this does not affect the assay. The extraction procedure can be proportionally scaled up if larger amount of sample is desired. Use 1-50 µl of the extracted sample per assay. If less than 50 µl of sample are used, top up with Assay Buffer until 50 µl. 7
9 2. Standard Curve Preparation: a. For the colorimetric assay: Add 0, 2, 4, 6, 8, 10 µl Palmitic Acid Standard into 96-well plate individually. Adjust volume to 50 µl/well with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of the Fatty Acid Standard. b. For the fluorometric assay: Dilute the Palmitic Acid Standard to 0.1 nmol/µl by adding 10 µl of the Standard to 90 µl of Assay Buffer, mix well. Add 0, 2, 4, 6, 8, 10 µl into each well individually. Adjust volume to 50 µl/well with Assay Buffer to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well of the Fatty Acid Standard. 3. Acyl-CoA Synthesis: Add 2 µl ACS Reagent into all standard and sample wells. Mix well; incubate the reaction at 37 C for 30 min. 4. Reaction Mix: Mix enough reagents for the number of assays and standard performed: For each well, prepare a total 50 µl Reaction Mix containing: Assay Buffer 44 µl Fatty Acid Probe 2 µl Enzyme Mix 2 µl Enhancer 2 µl Mix well. Add 50 µl of the Reaction Mix to each well containing 50 µl of the Standard or test samples. Incubate the reaction for 30 min at 37 C, protect from light. 8
10 5. Measure OD 570nm for colorimetric assay or fluorescence at Ex/Em = 535/590 nm in a micro-plate reader. 5. Data Analysis Subtract background value (the zero control) from all standard and sample readings. Plot standard curve: nmol/well vs. OD 570nm or fluorescence readings. Then apply the sample readings to the standard curve to obtain Fatty Acid amount in the sample wells. Fatty Acid Concentration = Fa / Sv (nmol/µl or mm) Where: Fa is the Fatty Acid amount (nmol) in the well obtained from standard curve. Sv is the sample volume (µl) added to the sample well. 9
11 Standard curve of Palmitic acid performed according to the kit instructions. 10
12 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
13 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
14 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 (technical@abcam.com) or phone (select contact us on for the phone number for your region). 13
15 14
16 UK, EU and ROW Tel: +44 (0) US, Canada and Latin America Tel: ABCAM (22226) China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. 15
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