ab65390 HDL and LDL/VLDL Cholesterol Assay kit

Transcription

1 ab65390 HDL and LDL/VLDL Cholesterol Assay kit (Colorimetric/Fluorometric) Instructions for Use For the rapid, sensitive and accurate measurement of HDL and LDL/VLDL Cholesterol in various samples. This product is for research use only and is not intended for diagnostic use. Version 4 Last Updated 13 January 2015

2 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 STANDARD PREPARATION SAMPLE PREPARATION 11 ASSAY PROCEDURE and DETECTION 12. ASSAY PROCEDURE and DETECTION 13 DATA ANALYSIS 13. CALCULATIONS TYPICAL DATA 18 RESOURCES 15. QUICK ASSAY PROCEDURE TROUBLESHOOTING FAQs INTERFERENCES NOTES 26 Discover more at 1

3 INTRODUCTION 1. BACKGROUND HDL and LDL/VLDL Cholesterol Quantification Assay Kit (colorimetric/fluorometric) (ab65390) provides a simple quantification method of HDL and LDL/VLDL after a convenient separation of HDL from LDL and VLDL (very low-density lipoprotein) in serum samples. In the assay, cholesterol oxidase specifically recognizes free cholesterol and produces products which react with probe to generate color (OD570 nm) and fluorescence (Ex/Em = 538/587 nm). Cholesterol esterase hydrolyzes cholesteryl ester into free cholesterol, therefore, cholesterol ester and free cholesterol can be detected separately in the presence and 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. Discover more at 2

4 INTRODUCTION 2. ASSAY SUMMARY Separate HDL and LDL/VLDL Prepare Standard Curve and samples Prepare and Add Reaction mix Measure absorbance (OD570 nm) or Fluorescence (Ex/Em = 538/587 nm) Discover more at 3

5 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 4

6 GENERAL INFORMATION 5. 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 2 µg/µl Cholesterol Standard 100 µl -20 C -20 C 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully perform this assay: MilliQ water or other type of double distilled water (ddh 2 O) PBS Microcentrifuge Pipettes and pipette tips 96 well plate: black plates (clear bottoms) for fluorometric assay; clear plates for colorimetric assay Colorimetric or fluorescent microplate reader equipped with filter for OD570 nm or Ex/Em = 535/587 nm (respectively) Heat block or water bath Vortex Dounce homogenizer or pestle (if using tissue) Orbital shaker Discover more at 5

7 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 6

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. 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. Ensure complete removal of all solutions and buffers from tubes or plates during wash 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 7

9 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. 9.2 Precipitation Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C. 9.3 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. 9.4 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. 9.5 Cholesterol Esterase: Reconstitute in 220 µl Cholesterol Assay Buffer. Aliquot esterase so that you have enough volume to perform the desired number of assays. Store aliquots at -20 C. 9.6 Enzyme Mix: Reconstitute in 220 µl Cholesterol Assay Buffer. Aliquot enzyme mix so that you have enough volume to perform the desired number of assays. Store aliquots at -20 C. Discover more at 8

10 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 For colorimetric assay Prepare a 240 µl 0.25 µg/µl Cholesterol Standard by diluting 30 µl Cholesterol Standard in 210 µl of Cholesterol Assay Buffer Using the 0.25 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 [Cholesterol] in well µg/well µg/well µg/well µg/well µg/well µg/well Each dilution has enough amount of standard to set up duplicate reading (2 x 50 µl). Discover more at 9

11 ASSAY PRE ASSAY PREPARATION 10.2 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 240 µl µg/µl Cholesterol Standard by diluting 30 µl Cholesterol Standard (from step ) in 210 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 [Cholesterol] in well µg/well µg/well µg/well µg/well µg/well µg/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 10

12 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. For TOTAL cholesterol quantification Total cholesterol quantification Skip Separation Step Separation of HDL and LDL/VLDL: 11.1 Liquid samples (serum, plasma and other biological fluids): 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 2000 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. Discover more at 11

13 ASSAY PRE ASSAY PREPARATION Precipitates are 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. If the supernatant is cloudy, the sample should be recentrifuged. 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. Discover more at 12

14 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 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) Reaction Mix: Prepare 50 µl of Reaction Mix for each reaction: COLORIMETRIC Component Total Cholesterol Reaction (µl) Mix Free Cholesterol Reaction Mix (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. To detect free cholesterol only, omit the Cholesterol Esterase in the reaction, and substitute with 2 μl of Assay Buffer. 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. Discover more at 13

15 ASSAY PRE ASSAY PROCEDURE and DETECTION Mix enough reagents for the number of assays 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 +1) FLUOROMETRIC Component Total Cholesterol Reaction (µl) Mix Free Cholesterol Reaction Mix (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. To detect free cholesterol only, omit the Cholesterol Esterase in the reaction, and substitute with 2 μl of Assay Buffer. 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. Mix enough reagents for the number of assays 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 +1) Discover more at 14

16 ASSAY PRE ASSAY PROCEDURE and DETECTION 12.3 Add 50 µl of Total Cholesterol Reaction Mix into 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 Cholestereol sample wells Mix and incubate at 37 C for 60 min protected from light Measure output on a microplate reader: - Colorimetric assay: measure OD570 nm. - Fluorometric assay: measure Ex/Em= 535/587 nm. Discover more at 15

17 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) 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) Extrapolate sample readings from the standard curve plotted using the following equation: A = ( Corrected absorbance (y intercept) Slope ) 13.6 Concentration of samples in the test samples is calculated as: Cholesterol Concentration = ( A V ) D Discover more at 16

18 ASSAY PRE DATA ANALYSIS Where: A = amount of sample cholesterol 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. 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. Discover more at 17

19 ASSAY PRE 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. Discover more at 18

20 ASSAY PRE DATA ANALYSIS 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 were measured following the kit protocol. Discover more at 19

21 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 (µl) Mix TOTAL / FREE Fluorometric Reaction (µl) Mix 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 Incubate plate at 37 C for 60 minutes protected from light. Measure plate at OD570nm for colorimetric assay or Ex/Em= 538/587 nm for fluorometric assay. Discover more at 20

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 Colorimeters: Clear plates Fluorometric: black wells/clear bottom plate Use PCA precipitation protocol for deproteinization Use Dounce homogenizer (increase number of strokes); observe for lysis under microscope 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 21

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 (< 5µL) 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 as to be in the linear range Discover more at 22

24 RESOURCES 17. 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 precipitaion buffer can potentially interfere in HPLC or LC-MS applications. 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 Discover more at 23

25 RESOURCES 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. Discover more at 24

26 RESOURCES 18. INTERFERENCES These chemicals or biological will cause interferences in this assay causing compromised results or complete failure: EDTA Sodium Fluoride (NaF) Discover more at 25

27 RESOURCES 19. NOTES Discover more at 26

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