ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric) Instructions for Use For the rapid, sensitive and accurate measurement of Triglyceride in various samples. This product is for research use only and is not intended for diagnostic use. Version 6 Last Updated 27 March 2015
Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY 3 GENERAL INFORMATION 3. PRECAUTIONS 4 4. STORAGE AND STABILITY 4 5. MATERIALS SUPPLIED 5 6. MATERIALS REQUIRED, NOT SUPPLIED 5 7. LIMITATIONS 6 8. TECHNICAL HINTS 7 ASSAY PREPARATION 9. REAGENT PREPARATION 8 10. STANDARD PREPARATION 10 11. SAMPLE PREPARATION 12 ASSAY PROCEDURE and DETECTION 12. ASSAY PROCEDURE and DETECTION 14 DATA ANALYSIS 13. CALCULATIONS 16 14. TYPICAL DATA 18 RESOURCES 15. QUICK ASSAY PROCEDURE 20 16. TROUBLESHOOTING 21 17. FAQ 23 18. INTERFERENCES 25 19. NOTES 26 Discover more at www.abcam.com 1
INTRODUCTION 1. BACKGROUND Triglyceride Quantification Assay Kit (Colorimetric/Fluorometric) (ab65336) provides a sensitive, easy assay to measure TG concentration in a variety of samples. In the assay, TG are converted to free fatty acids and glycerol. The glycerol is then oxidized to generate a product which reacts with the probe to generate color (spectrophotometry at λ = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The kit can detect 2 pmol-10 nmol (or 2 10000 pm range) of triglyceride in various samples. The kit also detects monoglycerides and diglycerides. Triglycerides (TG) are the main constituent of vegetable oil, animal fat, LDL and VLDL, and play an important role as transporters of fatty acids as well as serving as an energy source. TG are broken down into fatty acids and glycerol, after which both can serve as substrates for energy producing and metabolic pathways. High blood levels of TG are implicated in atherosclerosis, heart disease and stroke as well as in pancreatitis. Discover more at www.abcam.com 2
INTRODUCTION 2. ASSAY SUMMARY Standard curve preparation Sample preparation Add lipase and incubate at RT for 20 min Add reaction mix and incubate at RT for 60 min protected from light Measure optical density (OD570 nm) or fluorescence (Ex/Em = 535/590 nm) Discover more at www.abcam.com 3
GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit at -20ºC in the dark immediately upon receipt. Kit has a storage time of 1 year from receipt, providing components have not been reconstituted. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in section 5. Aliquot components in working volumes before storing at the recommended temperature. Reconstituted components are stable for 2 months. Discover more at www.abcam.com 4
GENERAL INFORMATION 5. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) Storage Condition (After Preparation) Triglyceride Assay Buffer 25 ml -20 C -20 C Triglyceride Probe (in DMSO, anhydrous) 200 μl -20 C -20 C Lipase (Lyophilized) 1 vial -20 C -20 C Triglyceride Enzyme Mix (Lyophilized) 1 vial -20 C -20 C 1 mm Triglyceride Standard 0.3 ml -20 C -20 C 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: MilliQ water or other type of double distilled water (ddh 2 O) Microcentrifuge Pipettes and pipette tips Colorimetric or fluorescent microplate reader equipped with filter for OD570 nm or Ex/Em = 535/587 nm (respectively) 96 well plate: black plates (clear bottoms) for fluorometric assay; clear plates for colorimetric assay Orbital shaker Heat block or water bath Vortex Dounce homogenizer or pestle (if using tissue) NP-40 Discover more at www.abcam.com 5
GENERAL INFORMATION 7. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures. Do not use kit or components if it has exceeded the expiration date on the kit labels. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. Discover more at www.abcam.com 6
GENERAL INFORMATION 8. TECHNICAL HINTS This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Keep enzymes, heat labile components and samples on ice during the assay. Make sure all buffers and solutions are at room temperature before starting the experiment. Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers. Avoid foaming or bubbles when mixing or reconstituting components. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Ensure plates are properly sealed or covered during incubation steps. Make sure you have the right type of plate for your detection method of choice. Make sure the heat block/water bath and microplate reader are switched on. Discover more at www.abcam.com 7
ASSAY PREPARATION 9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening. 9.1 Triglyceride Assay Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C. 9.2 Triglyceride Standard: Frozen storage may cause the triglyceride standard to separate from the aqueous phase. To re-dissolve, keep the cap tightly closed and place in a hot water bath (~80-100 C) for 1 min or until the standard looks cloudy, and then vortex for 30 sec, the standard should become clear. Repeat the heat and vortex one more time. The Triglyceride Standard is now completely in solution, and ready to use. Aliquot standard so that you have enough volume to perform the desired number of assays. Store aliquots at - 20 C. 9.3 Triglyceride Probe: Ready to use as supplied. Warm by placing in a 37 C bath for 1 5 minutes to thaw the DMSO solution before use. NOTE: DMSO tends to be solid when stored at -20 C, even when let at room temperature, so it needs to melt for few minutes at 37 C. Aliquot probe so that you have enough volume to perform the desired number of assays. Store at - 20 C protected from light and moisture. Once the probe is thawed, use within two months. 9.4 Triglyceride Enzyme mix: Reconstitute in 220 μl Triglyceride Assay Buffer. Keep on ice during the assay. Aliquot enzyme mix so that you have enough volume to perform the desired number of assays. Store aliquots at - 20 C. Use within two months. Discover more at www.abcam.com 8
ASSAY PRE ASSAY PREPARATION 9.5 Lipase: Reconstitute in 220 μl Triglyceride Assay Buffer. Keep on ice during the assay. Aliquot lipase so that you have enough volume to perform the desired number of assays. Store at - 20 C. Use within two months. Discover more at www.abcam.com 9
ASSAY PRE ASSAY PREPARATION 10.STANDARD PREPARATION Always prepare a fresh set of standards for every use. Diluted standard solution is unstable and must be used within 4 hours. 10.1 For the colorimetric assay 10.1.1 Prepare a 0.2 mm Triglyceride standard by diluting 100 µl of the 1 mm standard in 400 µl of Assay Buffer. 10.1.2 Using 0.2 mm Triglyceride standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes: Standard # Volume of Triglyceride Standard (µl) Assay Buffer (µl) Final volume in well (µl) End [Triglyceride] in well 1 0 150 50 0 nmol/well 2 30 120 50 2 nmol/well 3 60 90 50 4 nmol/well 4 90 60 50 6 nmol/wzell 5 120 30 50 8 nmol/well 6 150 0 50 10 nmol/well Each dilution has enough amount of standard to set up duplicate reading (2 x 50 µl). Discover more at www.abcam.com 10
ASSAY PRE ASSAY PREPARATION 10.2 For the flurometric assay: 10.2.1 Prepare a 0.2 mm Triglyceride standard by diluting 40 µl of the 1mM standard in 160 µl of Assay Buffer. 10.2.2 Prepare a 0.02 mm Triglyceride standard by diluting 50 µl of the 0.2mM Triglyceride standard with 450 µl of ddh2o. 10.2.3 Using 0.02 mm Triglyceride standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes: Standard # Volume of Triglyceride Standard (µl) Assay Buffer (µl) Final volume in well (µl) End [Triglyceride] in well 1 0 150 50 0 pmol/well 2 30 120 50 200 pmol/well 3 60 90 50 400 pmol/well 4 90 60 50 600 pmol/well 5 120 30 50 800 pmol/well 6 150 0 50 1 nmol/well Each dilution has enough amount of standard to set up duplicate reading (2 x 50 µl). NOTE: If your sample readings fall out the range of your fluorometric standard curve, you might need to adjust the dilutions and create a new standard curve. Discover more at www.abcam.com 11
ASSAY PRE ASSAY PREPARATION 11.SAMPLE PREPARATION General Sample information: We recommend performing several dilutions of your sample to ensure the readings are within the standard value range. We recommend that you use fresh samples. If you cannot perform the assay at the same time, we suggest that you complete the Sample Preparation step before storing the samples. Alternatively, if that is not possible, we suggest that you snap freeze cells or tissue in liquid nitrogen upon extraction and store the samples immediately at -80 C. When you are ready to test your samples, thaw them on ice. Be aware however that this might affect the stability of your samples and the readings can be lower than expected. 11.1 Cells (adherent or suspension) samples: 11.1.1 Harvest the amount of cells necessary for each assay (initial recommendation = 1 x 10 7 cells). 11.1.2 Wash cells with cold PBS. 11.1.3 Resuspend and homogenize samples in 1 ml of 5% NP- 40/ddH 2 O solution. 11.1.4 Slowly heat the samples to 80 100 C in a water bath for 2 5 minutes or until the NP-40 becomes cloudy, then cool down to room temperature. 11.1.5 Repeat previous step to solubilize all triglyceride. 11.1.6 Centrifuge for 2 minutes at top speed using a microcentrifuge to remove any insoluble material. 11.1.7 Dilute samples 10-fold with ddh 2 O before proceeding with the assay. 11.2 Tissue Samples: 11.2.1 Harvest the amount of tissue necessary for each assay (initial recommendation = 100 mg tissue). 11.2.2 Wash tissue in cold PBS. Discover more at www.abcam.com 12
ASSAY PRE ASSAY PREPARATION 11.2.3 Resuspend and homogenize in 1 ml of 5%NP-40/ddH 2 O solution using a Dounce homogenizer or pestle with 10 15 passes. 11.2.4 Slowly heat the samples to 80 100 C in a water bath for 2 5 minutes or until the NP-40 becomes cloudy, then cool down to room temperature. 11.2.5 Repeat the heating one more time to solubilize all triglyceride. 11.2.6 Centrifuge for 2 minutes at top speed using a microcentrifuge to remove any insoluble material. 11.2.7 Dilute samples 10-fold with ddh 2 O before proceeding with the assay. 11.3 Serum samples: Serum contains 0.1 6 mm triglyceride, which can be tested directly. NOTE: We suggest using different volumes of sample to ensure readings are within the Standard Curve range. Endogenous compounds in the sample may interfere with the reaction. To ensure accurate determinations of Triglyceride in our sample, we recommend spiking samples with a known amount of Standard (2 10 nmol). Discover more at www.abcam.com 13
ASSAY PROCEDURE and DETECTION 12. ASSAY PROCEDURE and DETECTION Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate. 12.1 Set up Reaction wells: - Standard wells = 50 µl standard dilutions. - Sample wells = 2 50 µl samples (adjust volume to 50 µl/well with Triglyceride Assay Buffer). - Sample background control wells = 2 50 µl samples (adjust volume to 50 µl/well with Triglyceride Assay Buffer). NOTE: for samples that contain glycerol, as it can interfere with the lipase activity. 12.2 Add Lipase: Add lipase to reactions as follows: - Standard wells = 2 µl Lipase. - Sample wells = 2 µl Lipase - Sample background wells = 2 µl Triglyceride Assay Buffer (do not add Lipase to these samples). Mix and incubate 20 minutes at room temperature to convert triglyceride to glycerol and fatty acid. 12.3 Triglyceride Reaction Mix: Prepare 50 µl of Reaction Mix for each reaction: Component Colorimetric Reaction (µl) Mix Fluorometric Reaction (µl) Triglyceride Assay buffer 46 47.6 Triglyceride Probe* 2 0.4 Triglyceride Enzyme Mix 2 2 Mix Discover more at www.abcam.com 14
ASSAY PRE ASSAY PREPARATION *For fluorometric readings, using 0.4 μl/well of the Triglyceride probe decreases the background readings, therefore increasing detection sensitivity. Mix enough reagents for the number of assays (samples, standards and background control) to be performed. Prepare a Master Mix of the Reaction Mix to ensure consistency. We recommend the following calculation: X µl component x (Number samples + standards + background control + 1) 12.4 Add 50 µl of Reaction Mix into each well. 12.5 Mix and incubate at room temperature for 60 minutes protected from light. 12.6 Measure assay in a suitable microplate reader: - Colorimetric assay: measure OD570nm - Fluorometric assay: measure Ex/Em = 535/590 nm. The reaction is stable for at least two hours. Discover more at www.abcam.com 15
DATA ANALYSIS 13. CALCULATIONS Samples producing signals greater than that of the highest standard should be further diluted in appropriate buffer and reanalyzed, then multiplying the concentration found by the appropriate dilution factor. For statistical reasons, we recommend each sample should be assayed with a minimum of two replicates (duplicates). 13.1 Average the duplicate reading for each standard and sample. 13.2 If sample background control is significant, then subtract the sample background control from the sample readings. 13.3 Subtract the mean absorbance value of the blank (Standard #1) from all standard and sample readings. This is the corrected absorbance. 13.4 Plot the corrected absorbance values for each standard as a function of the final concentration of Triglyceride. 13.5 Draw the best smooth curve through these points to construct the standard curve. Most plate reader software or Excel can plot these values and curve fit. Calculate the trendline equation based on your standard curve data (use the equation that provides the most accurate fit). 13.6 Extrapolate sample readings from the standard curve plotted using the following equation: Ts = ( Corrected absorbance (y intercept) Slope ) 13.7 Concentration of samples in the test samples is calculated as: Concentration = ( Sv) Ts D Where: Ts = amount of Triglyceride (nmol) from standard curve. Sv = volume of sample (µl) added in sample wells. D = sample dilution factor. Discover more at www.abcam.com 16
ASSAY PRE ASSAY PREPARATION Triglyceride molecular weight = 885.4 g/mol 13.8 For spiked samples, correct any sample interference by subtracting the sample from spiked sample reading. For spiked samples, the concentration of Triglyceride in sample well is calculated as: Sa = ( (ODs cor) (ODs + Sa cor) (ODs cor)) TGA Spike (nmol) Where: ODs cor = OD sample corrected. ODs = OD sample. Sa cor = TGA (triglyceride) amount from standard curve corrected. Discover more at www.abcam.com 17
ASSAY PRE ASSAY PREPARATION 14. TYPICAL DATA TYPICAL STANDARD CURVE Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed. Figure 1: Typical Triglyceride Standard calibration curve using colorimetric reading. Discover more at www.abcam.com 18
ASSAY PRE ASSAY PREPARATION Figure 2: Determination of Triglyceride in pooled normal human serum. Serum sample (3 µl) was spiked with a known amount of Triglyceride as internal Standard (4 nmol) and assayed according to the kit protocol. Discover more at www.abcam.com 19
RESOURCES 15.QUICK ASSAY PROCEDURE NOTE: This procedure is provided as a quick reference for experienced users. Follow the detailed procedure when performing the assay for the first time. Solubilize Triglyceride standard, thaw Triglyceride probe and prepare enzyme mix and lipase (aliquot if necessary); get equipment ready. Prepare appropriate standard curve for your detection method of choice (colorimetric or fluorometric). Prepare samples in optimal dilutions so that they fit standard curve readings. Set up plate in duplicate for standard (50 µl), samples (50 µl), and if appropriate, for sample background control wells (50 µl). Add 2 µl Lipase to standard and sample wells or 2 µl Assay Buffer to sample background control wells. Mix and incubate for 20 min at RT. Prepare 50 µl of appropriate Reaction Mix (Number samples + standards + background control + 1) for each reaction. Component Add 50 µl Triglyceride Reaction mix to standard, sample and sample background control wells. Incubate plate at RT for 60 min Colorimetric Reaction (µl) Measure plate at OD570 nm for colorimetric assay or Ex/Em = 535/590 nm for fluorometric assay. Mix Fluorometric Reaction Mix (µl) Triglyceride Assay buffer 46 47.6 Triglyceride Probe* 2 0.4 Triglyceride Enzyme Mix 2 2 Discover more at www.abcam.com 20
RESOURCES Discover more at www.abcam.com 21
RESOURCES 16. TROUBLESHOOTING Problem Cause Solution Assay not working Sample with erratic readings Lower/ Higher readings in samples and Standards Use of ice-cold buffer Plate read at incorrect wavelength Use of a different 96- well plate Samples not deproteinized (if indicated on protocol) Cells/tissue samples not homogenized completely Samples used after multiple free/ thaw cycles Use of old or inappropriately stored samples Presence of interfering substance in the sample Improperly thawed components Allowing reagents to sit for extended times on ice Incorrect incubation times or temperatures Buffers must be at room temperature Check the wavelength and filter settings of instrument Colorimetric: Clear plates Fluorometric: black wells/clear bottom plate Use PCA precipitation protocol for deproteinization Use Dounce homogenizer, increase number of strokes Aliquot and freeze samples if needed to use multiple times Use fresh samples or store at - 80 C (after snap freeze in liquid nitrogen) till use Check protocol for interfering substances; deproteinize samples Thaw all components completely and mix gently before use Always thaw and prepare fresh reaction mix before use Verify correct incubation times and temperatures in protocol Discover more at www.abcam.com 22
RESOURCES Problem Cause Solution Standard readings do not follow a linear pattern Unanticipated results Pipetting errors in standard or reaction mix Air bubbles formed in well Standard stock is at incorrect concentration Measured at incorrect wavelength Samples contain interfering substances Sample readings above/ below the linear range Avoid pipetting small volumes (< 5 µl) and prepare a master mix whenever possible Pipette gently against the wall of the tubes Always refer to dilutions described in the protocol Check equipment and filter setting Troubleshoot if it interferes with the kit Concentrate/ Dilute sample so it is within the linear range Discover more at www.abcam.com 23
RESOURCES 17. FAQ What can be done if the lysed cells are not dissolving? The amount of 5 % NP-40 in water used can be increased. Also, the temperature can be raised above 80 C to get the particles into solution, in addition to repeating the heating and cooling for 2 cycles. What could be the explanation for negative OD values but positive BODIPY staining in cells? The kit can detect 2 pmol 10 nmol (or 2 10000 µm range) of triglyceride. It could be that the BODIPY staining is showing total lipids in these cells but the amount of triglycerides is low. The fluorometric version of this assay is 10X more sensitive than the colorimetric one and could be chosen to see if the readings make sense. Also, it is crucial to check the instrument settings while measuring the samples. Use the correct filter for 570nm detection. How much Triglyceride is there is serum samples? Typical serum levels of Triglyceride can range between 0.1 6 mm, individual experimental findings may vary. Are the triglycerides concentrated on the surface layer of the supernatant or distributed in the whole supernatant after the boiling step? It is possible that triglycerides aggregate upon freezing and thawing while in an aqueous solution and stick to the walls of the tube which can skew the results. Also, there could be a layer of fat/oil after boiling. Once the sample is centrifuged, the liquid can be collected in a fresh tube and thoroughly vortexed so that when the sample is added to a well, a homogenous solution is pipetted. Discover more at www.abcam.com 24
RESOURCES Can less than 10 million cells be used for this assay? Less cells can be used, but the yield of triglycerides might be less. The number of cells depends on the amount of triglycerides in them. If less cells are used the volume of NP40-water can be scaled down proportionately. Discover more at www.abcam.com 25
RESOURCES 18. INTERFERENCES These chemicals or biological will cause interferences in this assay causing compromised results or complete failure. Sodium azide content above 0.05% Phenol red: typically does not interfere but when the color of the reaction in the well changes due to the amount used, then the red color can potentially interfere with the assay. Discover more at www.abcam.com 26
RESOURCES 19. NOTES Discover more at www.abcam.com 27
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