ab83360 Ammonia Assay Kit (Colorimetric)

Transcription

1 ab83360 Ammonia Assay Kit (Colorimetric) Instructions for Use For the rapid, sensitive and accurate measurement of ammonia levels in various samples. This product is for research use only and is not intended for diagnostic use. Version 5 Last Updated 14 April 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 6 ASSAY PREPARATION 9. REAGENT PREPARATION STANDARD PREPARATION SAMPLE PREPARATION 10 ASSAY PROCEDURE and DETECTION 12. ASSAY PROCEDURE and DETECTION 12 DATA ANALYSIS 13. CALCULATIONS TYPICAL DATA 15 RESOURCES 15. QUICK ASSAY PROCEDURE TROUBLESHOOTING FAQs INTERFERENCES NOTES 23 Discover more at 1

3 INTRODUCTION 1. BACKGROUND Ammonia Assay Kit (Colorimetric) (ab83360) provides a rapid, simple, sensitive, and reliable assay suitable for research and high throughput assay of Ammonia or Ammonium. In the assay, ammonia or ammonium is converted to a product that reacts with the OxiRed probe to generate color (λ max = 570 nm) which can be easily quantified by plate reader. The kit can detect 1 nmol (~20 µm) of ammonia or ammonium, much more sensitive than measuring NADPH based ammonia assay. Ammonia is an important source of nitrogen for living systems. Nitrogen is required for the synthesis of amino acids, which are the building blocks of protein. Ammonia is a metabolic product which is created through amino acid deamination. It plays an important role in both normal and abnormal animal physiology such as acid/base balance. Discover more at 2

4 INTRODUCTION 2. ASSAY SUMMARY Sample preparation Standard curve preparation Add reaction mix and incubate at 37 C for 60 min Measure optical density (OD570 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. All the solutions in this kit should be kept capped when not in use to prevent absorption of ammonia from the air. Discover more at 4

6 GENERAL INFORMATION 5. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) Storage Condition (After Preparation) Ammonia Assay Buffer 25 ml -20 C -20 C OxiRed Probe 200 µl -20 C -20 C Developer 1 vial -20 C -20 C Enzyme Mix (Lyophilized) 1 vial -20 C -20 C Converting Enzyme (Lyophilized) 1 vial -20 C -20 C NH 4 Cl Standard (10 mm) 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 utilize this assay: MilliQ water or other type of double distilled water (ddh 2 O) PBS Microcentrifuge Pipettes and pipette tips Colorimetric microplate reader equipped with filter for OD570 nm 96 well plate: clear plates for colorimetric assay Orbital shaker Dounce homogenizer or pestle (if using tissue) 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 and heat labile components and samples on ice during the assay. Make sure all buffers and developing solutions are at room temperature before starting the experiment. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Avoid foaming or bubbles when mixing or reconstituting components. Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers. 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 appropriate type of plate for the detection method of choice. Make sure the heat block/water bath and microplate reader are switched on before starting the experiment. Discover more at 7

9 ASSAY PREPARATION 9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening. All solutions in this kit should be kept tightly capped when not in use to prevent absorption of ammonia from the air. 9.1 Ammonia Assay Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C. 9.2 OxiRed 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 left at room temperature, so it needs to melt for few minutes at 37 C. Store at -20 C protected from light. Once the probe is thawed, use with two months. 9.3 Developer: Reconstitute in 220 µl Ammonia Assay Buffer. Aliquot developer so that you have enough volume to perform the desired number of assays Store at -20 C. Once reconstituted, use within 2 months. 9.4 Enzyme Mix: Reconstitute in 220 µl Ammonia Assay Buffer. Aliquot enzyme so that you have enough volume to perform the desired number of assays. Store at -20 C. Once reconstituted, use within 2 months. 9.5 Converting Enzyme: Reconstitute in 220 µl Ammonia Assay Buffer. Aliquot converting enzyme so that you have enough volume to perform the desired number of assays Store at -20 C. Once reconstituted, use within 2 months. 9.6 NH 4 Cl Standard: Ready to use as supplied. Aliquot standard so that you have enough volume to perform the desired number of assays. Store at -20 C. Keep on ice while in use. 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 Prepare 100 µl of 1 mm Ammonium Chloride Standard by adding 10 µl of the 10 mm Ammonium Chloride Standard to 90 µl of ddh 2 O Using 1 mm standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes: Standard # Volume of Standard (µl) Assay Buffer (µl) Final volume standard in well (µl) End [Ammonia] in well nmol/well nmol/well nmol/well nmol/well nmol/well nmol/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 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 Cell (adherent or suspension) samples: Harvest the amount of cells necessary for each assay (initial recommendation = 2 x 10 6 cells). (Equivalent of 1-5 x 10 4 cells/well required) Wash cells in cold PBS Resuspend cells in 100 µl Assay Buffer Homogenize cells quickly by pipetting up and down a few times Centrifuge sample for 2 5 minutes at 4 C at top speed using a cold microcentrifuge to remove any insoluble material Collect supernatant and transfer to a clean tube Keep on ice Tissue samples: Harvest the amount of tissue necessary for each assay (initial recommendation = 10 mg). (Equivalent of µg/well required). Discover more at 10

12 ASSAY PRE ASSAY PREPARATION Wash tissue in cold PBS Resuspend tissue in 100 µl Assay Buffer Homogenise tissue with a Dounce homogenizer sitting on ice, with passes Centrifuge samples for 2 5 minutes at 4 C at top speed using a cold microcentrifuge to remove any insoluble material Collect supernatant and transfer to a clean tube Keep on ice Plasma, Serum and Urine and biological fluids: Plasma, serum and urine can be tested directly in the assay. However, to find the optimal values and ensure your readings will fall within the standard values, we recommend performing several dilutions of the sample (1/2 1/5 1/10). NOTE: We recommend 5-15 µl/well of serum/plasma and <0.5 µl/well of urine. NOTE: We suggest using different volumes of sample to ensure readings are within the Standard Curve range. Discover more at 11

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. NOTE: Pyruvate in samples will interfere with the assay. If a significant amount of pyruvate is suspected in your samples, set up Sample Controls. The pyruvate reading must be subtracted from sample readings Set up Reaction wells: - Standard wells = 50 µl standard dilution. - Sample wells = 2 50 µl samples (adjust volume to 50 µl/well with Assay Buffer). - (Optional) Sample Control wells = 2 50 µl samples (adjust volume to 50 µl/well with Assay Buffer) Ammonia Reaction Mix: Prepare 50 µl of Reaction Mix for each reaction: Component Reaction Mix Samples (µl) Sample Control Reaction Mix (µl) Ammonia Assay Buffer OxiRed Probe 2 2 Enzyme Mix 2 2 Developer 2 2 Converting Enzyme 2 0 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 +1) Add 50 µl of the Reaction Mix to the standard and samples wells. Discover more at 12

14 ASSAY PRE ASSAY PROCEDURE and DETECTION 12.4 (Optional) Add 50 µl Sample Control Reaction Mix to Sample Control wells Mix and incubate at 37 C for 60 minutes protected from light Measure output on a microplate reader. - Colorimetric assay: measure OD570 nm. Discover more at 13

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) 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 NH 4 Cl 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: Sa = ( Corrected absorbance (y intercept) Slope ) 13.6 Concentration of samples in the test samples is calculated as: Ammonia or ammonium = ( Sa Sv) D Where: Sa = Sample amount (in nmol) from standard curve. Sv = Sample volume (µl) added into the wells. D = Sample dilution factor. NH 4 + Molecular Weight is g/mol. Discover more at 14

16 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 ammonium standard calibration curve using colorimetric reading. Discover more at 15

17 ASSAY PRE DATA ANALYSIS Figure 2: Ammonia measured in cell culture medium and control medium, background signal subtracted (duplicates +/- SD). Figure 3: Ammonia measured in mouse tissue lysates (mg of extracted protein), background signal subtracted (duplicates +/- SD). Discover more at 16

18 ASSAY PRE DATA ANALYSIS Figure 4: Ammonia measured in cell lysates, background signal subtracted (duplicates +/- SD). Figure 5: Ammonia measured in biological fluids, background signal subtracted (duplicates +/- SD). Discover more at 17

19 RESOURCES 15.QUICK ASSAY PROCEDURE NOTE: This procedure is provided as a quick reference for experienced users. Follow the detailed procedure when initially performing the assay. Solubilize enzyme mix, converting enzyme and developer, thaw OxiRed probe, NH 4 Cl standard and ammonia Assay Buffer (aliquot if necessary); get equipment ready Prepare standard curve Prepare samples in duplicate (find optimal dilutions to fit standard curve readings). Set up plate for standard (50 µl), samples (50 µl) and background wells (50 µl). Prepare Ammonia Reaction Mix and (Number samples + standards + 1) Component Reaction Mix Samples (µl) Sample Control Reaction Mix (µl) Ammonia Assay Buffer OxiRed Probe 2 2 Enzyme Mix 2 2 Developer 2 2 Converting Enzyme 2 0 Add 50 µl Reaction Mix to the standard and sample wells. (Optional) Add 50 µl Sample Control Reaction Mix to the control sample wells. Incubate at 37 C for 60 mins protected from light. Measure plate at OD570 nm for colorimetric assay. Discover more at 18

20 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 inappropriate plate for reader 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 Colorimetry: Clear plates Fluorescence: Black plates (clear bottom) 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 19

21 RESOURCES Problem Cause Solution Pipetting errors in standard or reaction mix Standard readings do not follow a linear pattern Unanticipated results 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 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 20

22 RESOURCES 17. FAQs Will ammonium will interfere with this assay (perhaps similar to pyruvate)? Yes. This kit measures the total ammonia and total ammonium concentration in your samples. Ammonium does not interfere with this assay. Discover more at 21

23 RESOURCES 18. INTERFERENCES These chemicals or biological materials will cause interferences in this assay causing compromised results or complete failure: Pyruvate if significant amount of pyruvate is suspected in your sample, set up a Sample control as in Section 12. Discover more at 22

24 RESOURCES 19. NOTES Discover more at 23

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