ab65390 HDL and LDL/VLDL Cholesterol Assay kit (Colorimetric/ Fluorometric)

Transcription

1 ab65390 HDL and LDL/VLDL Cholesterol Assay kit (Colorimetric/ Fluorometric) Instructions for use: For sensitive and accurate measurement of HDL and LDL/VLDL cholesterol in serum samples. This product is for research use only and is not intended for diagnostic use. Version 7 Last Updated 27 January 2016

2 Table of Contents INTRODUCTION 1 1. BACKGROUND 1 2. ASSAY SUMMARY 2 GENERAL INFORMATION 3 3. PRECAUTIONS 3 4. STORAGE AND STABILITY 3 5. LIMITATIONS 4 6. MATERIALS SUPPLIED 4 7. MATERIALS REQUIRED, NOT SUPPLIED 5 8. TECHNICAL HINTS 6 ASSAY PREPARATION 7 9. REAGENT PREPARATION STANDARD PREPARATION SAMPLE PREPARATION 10 ASSAY PROCEDURE ASSAY PROCEDURE 12 DATA ANALYSIS CALCULATIONS TYPICAL DATA 17 RESOURCES QUICK ASSAY PROCEDURE TROUBLESHOOTING INTERFERENCES FAQS NOTES 24

3 INTRODUCTION INTRODUCTION 1. BACKGROUND HDL and LDL/VLDL Cholesterol Assay Kit (Colorimetric/Fluorometric) (ab65390) provides a simple quantification method of HDL and LDL/VLDL after a convenient separation of the different lipoprotein fractions [HDL from LDL and VLDL (very low-density lipoprotein)] in serum samples. In this assay, cholesterol oxidase specifically recognizes free cholesterol and produces a component that will react with a probe to generate color (OD 570 nm) and fluorescence (Ex/Em = 538/587 nm). Because cholesterol esterase is able to hydrolyze cholesteryl ester into free cholesterol, both cholesterol ester and free cholesterol can be detected separately, depending on the presence or absence of cholesterol esterase in the reactions. Regulation of HDL (high-density-lipoprotein)-cholesterol and LDL (lowdensity-lipoprotein)-cholesterol plays a central role in various disease developments. It is well known that low levels of HDL and high level of LDL are associated with an increased risk of cardiovascular events. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 1

4 INTRODUCTION 2. ASSAY SUMMARY Separate HDL and LDL/VLDL Prepare standard curve Prepare samples Prepare and add reaction mix Measure absorbance (OD570 nm) or fluorescence (Ex/Em = 538/587 nm) ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 2

5 GENERAL INFORMATION 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. We understand that, occasionally, experimental protocols might need to be modified to meet unique experimental circumstances. However, we cannot guarantee the performance of the product outside the conditions detailed in this protocol booklet. Reagents should be treated as possible mutagens and should be handle with care and disposed of properly. Please review the Safety Datasheet (SDS) provided with the product for information on the specific components. Observe good laboratory practices. Gloves, lab coat, and protective eyewear should always be worn. Never pipet by mouth. Do not eat, drink or smoke in the laboratory areas. All biological materials should be treated as potentially hazardous and handled as such. They should be disposed of in accordance with established safety procedures. 4. STORAGE AND STABILITY Store kit at -20 C 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 the Materials Supplied Section. Aliquot components in working volumes before storing at the recommended temperature. Reconstituted components are stable for 2 months. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 3

6 GENERAL INFORMATION 5. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures. 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. 6. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) Storage Condition (After Preparation) Cholesterol Assay Buffer 25 ml -20 C -20 C 2X LDL/VLDL Precipitation Buffer 10 ml -20 C -20 C Cholesterol Probe (in DMSO, anhydrous) 200 µl -20 C -20 C Enzyme Mix (Lyophilized) 1 vial -20 C -20 C Cholesterol Esterase (Lyophilized) 1 vial -20 C -20 C Cholesterol Standard (2 µg/µl) 100 µl -20 C -20 C ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 4

7 GENERAL INFORMATION 7. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully perform this assay: Microplate reader capable of measuring absorbance at OD 570 nm or fluorescence at Ex/Em = 535/587 nm MilliQ water or other type of double distilled water (ddh 2 O) Pipettes and pipette tips, including multi-channel pipette Assorted glassware for the preparation of reagents and buffer solutions Tubes for the preparation of reagents and buffer solutions 96 well plate with clear flat bottom (for colorimetric assay) / 96 well plate with clear flat bottom, preferably black (for fluorometric assay) ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 5

8 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. Selected components in this kit are supplied in surplus amount to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. They should be disposed of in accordance with established safety procedures. 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. Ensure all reagents and solutions are at the appropriate temperature before starting the assay. Samples which generate values that are greater than the most concentrated standard should be further diluted in the appropriate sample dilution buffer. Make sure you have the right type of plate for your detection method of choice. Make sure all necessary equipment is switched on and set at the appropriate temperature. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 6

9 ASSAY PREPARATION ASSAY PREPARATION 9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening 9.1. Cholesterol Assay Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C Precipitation Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C Cholesterol Standard: Ready to use as supplied. Equilibrate to room temperature before use. Aliquot standard so that you have enough volume to perform the desired number of assays. Store at -20 C Cholesterol 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. Use within two months Cholesterol Esterase: Reconstitute in 220 µl Cholesterol Assay Buffer. Keep on ice during the assay. Aliquot esterase so that you have enough volume to perform the desired number of assays. Store aliquots at -20 C Enzyme Mix: Reconstitute in 220 µl Cholesterol 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. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 7

10 ASSAY PREPARATION 10.STANDARD PREPARATION Always prepare a fresh set of standards for every use. Discard the working standard dilutions after use as they do not store well For colorimetric assay Prepare a 250 µl 0.2 µg/µl Cholesterol Standard by diluting 25 µl Cholesterol Standard in 225 µl of Cholesterol Assay Buffer Using the 0.2 µg/µl Cholesterol Standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes: Standard # Volume of Cholesterol standard (µl) Assay Buffer (µl) Final volume standard in well (µl) End Conc Cholesterol in well (µg/well) Each dilution has enough amount of standard to set up duplicate reading (2 x 50 µl). ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 8

11 ASSAY PREPARATION For fluorometric assay Prepare a 0.25 µg/µl Cholesterol standard by diluting 5 µl Cholesterol Standard in 35 µl of Cholesterol Assay Buffer Prepare a 250 µl µg/µl Cholesterol Standard by diluting 25 µl Cholesterol Standard in 225 µl of Cholesterol Assay Buffer Using µg/µl Cholesterol Standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes: Standard # Volume of Cholesterol standard (µl) Assay Buffer (µl) Final volume standard in well (µl) End Conc Cholesterol in well (µg/well) Each dilution has enough amount of standard to set up duplicate reading (2 x 50 µl). ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 9

12 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 your samples in liquid nitrogen upon extraction and store them 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 Biological fluids (Serum and Plasma): Quantification of TOTAL CHOLESTEROL: Use samples directly; no preparation step is required. Proceed to Section 12. Separation of HDL and LDL/VLDL: Mix 100 µl of sample with 100 µl of 2X Precipitation Buffer in microcentrifuge tubes Incubate 10 minutes at room temperature Centrifuge at 2,000 x g (5,000 rpm on a bench-top microcentrifuge) for 10 minutes Transfer the supernatant into new labeled tubes. This is the HDL fraction Precipitates contain the LDL/VLDL fraction. To measure the LDL/VLDL fraction, centrifuge the precipitate again as in step Remove trace amount of HDL supernatant carefully Resuspend the precipitate in 200 µl PBS. This is the LDL/VLDL fraction. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 10

13 ASSAY PREPARATION If the supernatant is cloudy, the sample should be re-centrifuged. If the sample remains cloudy, dilute the sample 1:1 with PBS and repeat the separation procedure from step For serum and plasma: use 1 20 µl of the fractions. Adjust total volume to 50 µl with Cholesterol Assay Buffer. NOTE: We suggest using different volumes of sample to ensure readings are within the Standard Curve range. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 11

14 ASSAY PROCEDURE ASSAY PROCEDURE 12.ASSAY PROCEDURE Equilibrate all materials and prepared reagents to room temperature prior to use. We recommend that you assay all standards, controls and samples in duplicate. Prepare all reagents, working standards and samples as directed in the previous sections Set up Reaction wells: Standard wells = 50 µl Standard dilutions. Sample wells for TOTAL cholesterol = 2 50 µl samples (adjust volume to 50 µl/well with Assay Buffer). Sample wells for FREE cholesterol = 2 50 µl samples (adjust volume to 50 µl/well with Assay Buffer) Cholesterol Reaction Mix (COLORIMETRIC ASSAY): Prepare 50 µl of Reaction Mix for each reaction. Mix enough reagents for the number of assays (samples and controls) to be performed. Prepare a master of the Reaction Mix to ensure consistency. We recommend the following calculation: X µl component x (Number reactions +1) Component Total Cholesterol Free Cholesterol Reaction Mix Reaction Mix (µl) (µl) Cholesterol Assay Buffer Cholesterol Probe 2 2 Enzyme Mix 2 2 Cholesterol Esterase* 2 0 *NOTE: Cholesterol Esterase hydrolyzes cholesteryl ester to free cholesterol. If no esterase is added to the reaction, the assay only detects free cholesterol only. With the addition of Cholesterol Esterase, the assay detects total cholesterol (cholesterol and cholesteryl esters).cholesterol Esterase must be added to the standard curve wells to convert all the cholesterol in the standard solution. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 12

15 ASSAY PROCEDURE Add 50 µl of Total Cholesterol Reaction Mix to Standard wells Add 50 µl of Total Cholesterol Reaction Mix to Total Cholesterol sample wells Add 50 µl of Free Cholesterol Reaction Mix to Free Cholesterol sample wells Mix and incubate at 37 C for 60 minutes protected from light Measure output immediately on a microplate reader at OD 570 nm Cholesterol Reaction Mix (FLUOROMETRIC ASSAY) Prepare 50 µl of Reaction Mix for each reaction. Mix enough reagents for the number of assays (samples and controls) to be performed. Prepare a master of the Reaction Mix to ensure consistency. We recommend the following calculation: X µl component x (Number reactions +1) Component Total Cholesterol Free Cholesterol Reaction Mix Reaction Mix (µl) (µl) Cholesterol Assay Buffer Cholesterol Probe Enzyme Mix 2 2 Cholesterol Esterase* 2 0 NOTE: For the fluorometric assay, use 0.4 μl/well of the Probe to decrease the background readings, therefore increasing detection sensitivity. *NOTE: Cholesterol Esterase hydrolyzes cholesteryl ester to free cholesterol. If no esterase is added to the reaction, the assay only detects free cholesterol only. With the addition of Cholesterol Esterase, the assay detects total cholesterol (cholesterol and cholesteryl esters).cholesterol Esterase must be added to the standard curve wells to convert all the cholesterol in the standard solution. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 13

16 ASSAY PROCEDURE Add 50 µl of Total Cholesterol Reaction Mix to Standard wells Add 50 µl of Total Cholesterol Reaction Mix to Total Cholesterol sample wells Add 50 µl of Free Cholesterol Reaction Mix to Free Cholesterol sample wells Mix and incubate at 37 C for 60 minutes protected from light Measure output immediately on a microplate reader at Ex/Em= 535/587 nm. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 14

17 DATA ANALYSIS DATA ANALYSIS 13.CALCULATIONS Samples producing signals greater than that of the highest standard should be further diluted in appropriate buffer and reanalyzed, then multiply the concentration found by the appropriate dilution factor Average the duplicate reading for each standard and sample Subtract the mean absorbance value of the blank (Standard #1) from all standard and sample readings. This is the corrected absorbance Plot the corrected absorbance values for each standard as a function of the final concentration of Cholesterol 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) Concentration of Cholesterol in the test samples is calculated as: Cholesterol Concentration = ( A V) D Where: A = amount of cholesterol in the sample well calculated from the standard curve (µg). V = volume of sample added to the sample reaction well (µl). D = Dilution Factor. For Total cholesterol, D = 1; for HDL and LDL/VLDL fractions, D = 2. Cholesterol Molecular Weight: g/mol 1 µg/µl = 100 mg/dl. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 15

18 DATA ANALYSIS Total Cholesterol (Free Cholesterol + Cholesteryl esters): use Total Cholesterol Reaction Mix. Free Colesterol: use Free Cholesterol Reaction Mix. Cholesteryl esters: Total Cholesterol value Free Cholesterol value. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 16

19 DATA ANALYSIS 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 Cholesterol Standard calibration curve using colorimetric assay. Figure 2: Measurement of total cholesterol, HDL, LDL/VLDL from serum samples from various species. Total Cholesterol (blue bar), HDL (green bar), and LDL/VLDL (yellow bar) cholesterol. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 17

20 DATA ANALYSIS Figure 3: Wild type (WT) or farnesoid X receptor knock-out (FRX-KO) mice were treated with either vehicle (Veh) or a high fat-containing diet (HFD) for 16 weeks. Serum HDL (top) and serum LDL/VLDL cholesterol (bottom) from 5-7 mice/group was measured using ab65390 following protocol instructions. An asterisk (*) means P<0.05 between WT and FXR-KO vehicle group. A pound (#) indicates P<0.05 between WT and FXR-KO HFD group. Image obtained from Li G et al., PLoS One, 2012; 7(4): e35895 ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 18

21 RESOURCES 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 Cholesterol standard, thaw Cholesterol probe, prepare enzyme mix and Cholesterol Esterase (aliquot if necessary); get equipment ready. Prepare appropriate standard curve for your detection method of choice (colorimetric or fluorometric). Prepare samples in duplicate (find optimal dilutions to fit standard curve readings). Prepare Total Cholesterol + Free Cholesterol Reaction Mixes (Number samples+standards+1) for colorimetric or fluorometric reading. Component Colorimetric Reaction Mix (µl) TOTAL / FREE Fluorometric Reaction Mix (µl) TOTAL / FREE Cholesterol Assay Buffer 44 / / 47.6 Cholesterol Probe 2 / / 0.4 Enzyme Mix 2 / 2 2 / 2 Cholesterol Esterase 2 / 0 2 / 0 Add 50 µl Reaction Mix to appropriate wells. Incubate plate at 37 C for 60 minutes protected from light. Measure plate immediately at OD 570 nm for colorimetric assay or Ex/Em= 538/587 nm for fluorometric assay. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 19

22 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 provided 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 ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 20

23 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 on protocol Check equipment and filter setting Troubleshoot if it interferes with the kit Concentrate/ Dilute sample so it is within the linear range ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 21

24 RESOURCES 17.INTERFERENCES These chemicals or biological materials will cause interferences in this assay causing compromised results or complete failure: EDTA Sodium Fluoride (NaF) 18. FAQs Can tissues be used with this kit? Finely minced tissues can be homogenized using the assay buffer and then the same protocol can be followed as that for serum for HDL/LDL separation. Which anticoagulant should be used to collect blood to be used with this kit? Any other anticoagulant but EDTA is fine. Ideally Heparin would be the anti-coagulant of choice. Why is EDTA-plasma not ideal for this assay? EDTA is not ideal for measurement of HDL/LDL in samples. EDTA plasma has potential disadvantages that have discouraged its use. Inadequate mixing with EDTA can result in microclots. Also, EDTA osmotically draws water from red cells, diluting the plasma constituents. Can the separated HDl/LDL/VLDL be used for LC-MS or HPLC? We have not tested the components in an HPLC system. This is optimized as a cell based assay in a plate format. Components in the precipitation buffer can potentially interfere in HPLC or LC-MS applications. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 22

25 RESOURCES Why is there higher free LDL than total LDL in some samples? There are couple of possibilities for that phenomenon: 1. After treatment with the esterase, the free cholesterol in these samples is found associated to membrane/lipid, thus making it difficult to detect. 2. The cholesterol esters can be associated with serum lipids in the samples and therefore be inaccessible for the esterase to convert them to free cholesterol. Adding a small volume of detergent to solubilize the lipids might help. This could increase the reading for the total cholesterol. Add NP-40 at 0.05% concentration in the assay buffer and then mix with the sample before adding to each well; the idea being to keep the lipids solubilized and not in rafts/other lipid structures trapping the cholesterol in them. Additionally, if there is any sodium fluoride (NaF) in the sample (NaF being frequently used as a phosphatase inhibitor), it can inhibit the cholesterol esterase which could lead to these results. ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 23

26 RESOURCES 19.NOTES ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 24

27 RESOURCES ab65390 HDL and LDL/VLDL Cholesterol Assay Kit 25

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