Version 1 Last updated 25 May 2018 ab208559 Homocysteine Assay Kit (Fluorometric) For the measurement of homocysteine in mammalian plasma and serum. This product is for research use only and is not intended for diagnostic use. ab208559 Homocysteine Assay Kit (Fluorometric) 1
Table of Contents 1. Overview 3 2. Materials Supplied and Storage 4 3. Materials Required, Not Supplied 5 4. General guidelines, precautions, and troubleshooting 6 5. Reagent Preparation 7 6. Standard Preparation 8 7. Sample Preparation 9 8. Assay Procedure 10 9. Data Analysis 12 10. Typical Data 13 11. Notes 14 ab208559 Homocysteine Assay Kit (Fluorometric) 2
1. Overview Homocysteine Assay Kit (Fluorometric) (ab208559) is a simple, highly sensitive, high-throughput screening adaptable assay to measure Homocysteine in mammalian plasma and serum. The reduction of homocysteine disulphides to free homocysteine, which is cleaved by a homocysteine-selective enzyme, generates an intermediate which reacts with a probe solution to create a stable fluorophore that emits in the far-red spectrum (Ex/Em = 658/708 nm). This assay can detect homocysteine levels as low as 5 µm. The kit provides enough reagents for 100 assays using the methods as described. Add sample to plate well Add Homocysteine Assay Buffer with Disulphide Reducing Agent to each sample Incubate at 37 C for 30 minutes with gentle shaking Add Homocysteine Enzyme Mix to each sample, incubate at room temperature for 5 minutes, protected from light Add Fluorogenic Developer Mix, incubate for 15 minutes with continuous shaking and measure fluorescence at Ex/Em = 658/708 in endpoint mode ab208559 Homocysteine Assay Kit (Fluorometric) 3
2. Materials Supplied and Storage Store kit at -20 C in the dark immediately on receipt and check below for storage for individual components. Kit can be stored for 1 year from receipt, if components have not been reconstituted. Aliquot components in working volumes before storing at the recommended temperature. Item Quantity Storage temperatur e (before prep) Storage temperatur e (after prep) Homocysteine Assay Buffer 25 ml -20 C -20 C Disulfide Reducing Agent (DTT) 300 µl -20 C Homocysteine Enzyme Mix 1 vial -20 C -20 C (avoid repeated freeze-thaws) -20 C (avoid repeated freeze-thaws) Fluorogenic Probe Solution 5 ml -20 C 4 C Developer Solution 5 ml -20 C 4 C Homocysteine Disulfide Standard 1 vial -20 C -20 C (stable for 5 freeze/thaw cycles) ab208559 Homocysteine Assay Kit (Fluorometric) 4
3. 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 fluorescence at Ex/Em = 658/708 nm White 96 well plate with flat bottom. (Optional) Protease inhibitors: we recommend Protease Inhibitor Cocktail II (ab201116) [AEBSF, aprotinin, E-64, EDTA, leupeptin] as general use cocktail. ab208559 Homocysteine Assay Kit (Fluorometric) 5
4. General guidelines, precautions, and troubleshooting Please observe safe laboratory practice and consult the safety datasheet. For general guidelines, precautions, limitations on the use of our assay kits and general assay troubleshooting tips, particularly for first time users, please consult our guide: www.abcam.com/assaykitguidelines For typical data produced using the assay, please see the assay kit datasheet on our website. ab208559 Homocysteine Assay Kit (Fluorometric) 6
5. Reagent Preparation Briefly centrifuge small vials at low speed prior to opening. 5.1 Homocyteine Assay Buffer: Ready to use as supplied. Aliquot enough Homocysteine Assay Buffer for the number of reactions to be performed and warm to room temperature prior to use. 5.2 Disulfide Reducing Agent (DTT) Provided as a 100 mm stock solution. Aliquot and store at - 20 C, avoid repeated freeze/thaw cycles. Prepare a working stock solution by adding Disulfide Reducing Agent (DTT) to Homocysteine Assay Buffer at a 1:100 ratio (10 µl of 100 mm DTT stock per 1 ml of Homocysteine Assay Buffer) immediately prior to use. 5.3 Homocysteine Enzyme Mix Reconstitute in 330 µl of Homocysteine Assay Buffer to generate a 10X stock solution. Aliquot and store at -20 C, protected from light. Avoid repeated freeze/thaw cycles. Prepare a 1X Homocysteine Enzyme Mix solution by diluting the reconstituted 10X stock with Homocysteine Assay Buffer (without DTT) at a 1:10 ratio. 5.4 Fluorogenic Probe Solution Warm to room temperature prior to use. Store at 4 C, protected from light. 5.5 Developer Solution Warm to room temperature prior to use. Store at 4 C, protected from light. 5.6 Homocysteine Disulfide Standard Reconstitute in 220 µl of dh2o for a 1 mm solution. Store at - 20 C, stable for 5 freeze/thaw cycles. ab208559 Homocysteine Assay Kit (Fluorometric) 7
6. Standard Preparation Always prepare a fresh set of standards for every use. Discard working standard dilutions after use as they do not store well. 1. Prepare 400 µl of 25 µm Homocysteine Disulfide Standard by adding 10 µl of the 1 mm Homocysteine Disulfide stock to 390 µl of Homocysteine Assay Buffer. 2. Using 25 µm Homocysteine Disulfide Standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes: Standard # Homocysteine Disulfide Standard (µl) Assay Buffer (µl) Final volume standard in well (µl) End amount of Homocysteine Disulfide Standard in well (pmol/well) 1 0 170 170 0 2 1 169 170 50 3 2 168 170 100 4 4 166 170 200 5 6 164 170 300 6 8 162 170 400 7 10 160 170 500 ab208559 Homocysteine Assay Kit (Fluorometric) 8
7. 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 for the most reproducible assay. If you cannot perform the assay at the same time, 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. Avoid multiple freeze-thaws. For certain samples, it may be advantageous to add protease inhibitors: we recommend Protease Inhibitor Cocktail II (ab201116) [AEBSF, aprotinin, E-64, EDTA, leupeptin] as a general use cocktail. ab208559 Homocysteine Assay Kit (Fluorometric) 9
8. Assay Procedure Equilibrate all materials and prepared reagents to room temperature just prior to use and gently agitate. Assay all standards, controls and samples in duplicate. 8.1 Preliminary 1. Prepare sample buffer (section 5.2) 2. Prepare Homocysteine Enzyme Mix (Section 5.3). 3. Prepare Homocysteine Disulphide Standard (Section 5.6). 8.2 Reaction wells set up: Standard wells = 170 µl standard dilutions. Sample wells = 10 µl samples (adjust volume to 170 µl/well with Homocysteine Assay Buffer). Sample Background Control wells = 10 µl samples (adjust volume to 170 µl/well with Homocysteine Assay Buffer). 8.3 Homocysteine Reaction mix: 1. Prepare 30 µl of Homocysteine Reaction Mix and Background Mix for each reaction. Prepare a master mix to ensure consistency. Component Homocysteine Assay Buffer (without DTT) Reaction Mix (µl) Background Reaction Mix (µl) 27 30 1X Homocysteine Enzyme Mix 3 0 2. Add 30 µl of Reaction Mix into each standard and sample wells. 3. Add 30 µl of Background Reaction Mix into the background control sample wells. 4. Mix well and incubate the plate at room temperature for 5 mins, protected from light. 5. During the 5 min incubation period, prepare enough Fluorogenic Developer Mix for the number of reactions being performed. For each reaction well, mix 30 µl Fluorogenic Probe Solution and 20 ab208559 Homocysteine Assay Kit (Fluorometric) 10
µl Developer Solution. Add 50 µl Fluorogenic Developer Mix to all sample, background control and standard curve wells and mix well (bringing the final volume to 250 µl/well). 6. Following addition of Fluorogenic Developer Mix, incubate plate for 15 min with continuous shaking (to ensure adequate mixing). Measure the fluorescence of all sample, background and standard curve wells at Ex/Em = 658/708 nm in endpoint mode. ab208559 Homocysteine Assay Kit (Fluorometric) 11
9. Data Analysis 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. 1. Average the duplicate reading for each standard, control and sample. 2. Subtract the mean value of the blank (Standard #1) from all standards, controls and sample readings. This is the corrected absorbance. 3. If significant, subtract the sample background control from sample readings. 4. Plot the corrected values for each standard as a function of the final concentration of Homocysteine. 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). 6. Apply the corrected sample O.D. reading to the standard curve to get Homocysteine (B) amount in the sample wells. 7. Concentration of Homocysteine in µm in the test samples is calculated as: Homocysteine concentration = B V * D Where: B = amount of Homocysteine in the sample well calculated from standard curve in µm V = sample volume added in the sample wells (10 µl). D = sample dilution factor if sample is diluted to fit within the standard curve range (prior to reaction well set up). ab208559 Homocysteine Assay Kit (Fluorometric) 12
10. Typical Data Data provided for demonstration purposes only. Figure 1. Typical Homocysteine standard curve and assay data. (a) Homocysteine standard curve. (b) Specificity for detection of Homocysteine (HCY) over other thiols. At an 8-fold molar excess versus HCY, cysteine (CYS) contributes 15% interference, while methionine (MET) and glutathione (GSH) contribute 2%. (c) Estimation of total HCY in single-donor human plasma and serum (10 µl), spiked with 50 pmol Homocysteine Disulphide Standard (equivalent to 100 pmol or 10 µm free HCY). Total HCY concentrations for plasma and serum samples were 10.1 ± 0.28 µm and 15.9 ± 0.99 µm, with respective spike recoveries of 95.1% and 103.4%. Data are mean ± SEM of 3 replicates, assayed according to the kit protocol. ab208559 Homocysteine Assay Kit (Fluorometric) 13
11. Notes ab208559 Homocysteine Assay Kit (Fluorometric) 14
ab208559 Homocysteine Assay Kit (Fluorometric) 15
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