Nitric Oxide Colorimetric Detection Kit - B
Contents
1. 96 well plate reader capable of reading optical absorption at 540 570 nm Software for converting optical density OD readings from the plate reader and carrying out four parameter logistic curve 4PLC fitting Contact your plate reader manufacturer for details PRECAUTIONS For in vitro research use only As with all such products this kit should only be used by qualified personnel who have had laboratory safety instruction The complete insert should be read and understood before attempting to use the product The Color Reagents A and B are both acid solutions and should be handled like any laboratory acid Wear gloves and laboratory coats when handling materials and in all cases please consult your institution s safety procedures for working with hazardous chemicals B Bridge International Inc Nitric Oxide 041812 REAGENT PREPARATION Allow the kit reagents to come to room temperature for 30 minutes Allow the kit reagents to come to room temperature for 30 minutes We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine NO concentrations Ensure that all samples have reached room temperature and have been diluted and filtered through a 10 000 MWCO filter prior to running them in the kit Nitrate Reductase NR Allow the desiccator to warm to room temperature Add 550 uL of Enzyme Stabilization Buffer to the vial Vortex gently and allow to sit at room temperature for 5 m2. B Bridge International Inc wA Nitric Oxide Colorimetric Detection Kit User Manual Catalog K3023 1 B Bridge International Inc Nitric Oxide 041812 TABLE OF CONTENTS Intended Use Background Assay Principle Kit Components Materials Required Precautions Reagent Preparation Sample Preparation Assay Protocol Calculations Typical Data Example CONN OD Oo fF FP A OOO Typical Standard Curve Example Notice to Purchaser This product is to be used for Research Purposes Only It is not to be used for Drug or Diagnostic Purposes nor is it intended for Human Use B Bridge products may not be resold modified for resale or used to manufacture commercial products without the express written consent of B Bridge International Inc EXCEPT AS OTHERWISE EXPRESSLY SET FORTH IN THIS USER MANUAL B BRIDGE DOES NOT MAKE ANY REPRESENTATION OR WARRANTIES OR CONDITIONS OF ANY KIND EITHER EXPRESSED OR IMPLIED WITH RESPECT TO THE PRODUCTS OR INFORMATION DISCLOSED HEREUNDER INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FIT FOR A PARTICULAR PURPOSE OR NONINFRINGEMENT OF THE INTELLECTUAL PROPERTY RIGHTS OF THIRD PARTIES B Bridge International Inc All Rights Reserved 2 B Bridge International Inc Nitric Oxide 041812 INTENDED USE The B Bridge Nitric Oxide Detection Kit catalog K3023 1 quantitatively measure Nitrate and Nitrite present in serum plasma urine and saliva as well as water and b
3. acturer s recommendations Collect the filtrates and either further dilute with Assay Buffer as appropriate or use directly in the assay For serum and plasma the recommended final dilution is 21 4 For urine and saliva the recommended final dilution is 21 8 ASSAY PROTOCOL Standards and samples should be run in duplicate Use the appropriate standards for either Nitrite NO or Nitrate NO3 determination All samples should be diluted and filtered through a 10 000 MWCO filter prior to using Nitrite Determination Protocol 1 Pipet 50 uL of samples or Nitrite standards into duplicate wells in the plate 2 Pipet 50 uL of Assay Buffer into duplicate wells as the Zero standard 3 Add 25 uL of the Color Reagent A to each well using a repeater pipet 4 Add 25 uL of the Color Reagent B to each of well using a repeater pipet 5 Incubate at room temperature for 5 minutes 6 Read the optical density at 540 570 nm These readings are for the Nitrite determination Total Nitric Oxide Determination Protocol 1 Pipet 50 uL of samples or Nitrate standards into duplicate wells in the plate 2 Pipet 50 uL of Assay Buffer into duplicate wells as the Zero standard 3 Add 10 uL of prepared NADH to each well using a repeater pipet 4 Add 10 uL of prepared NR to each well using a repeater pipet 5 Incubate at room temperature for 20 minutes 6 Add 25 uL of the Color Reagent A to each well using a repeater pipet 7 Add 25 uL of the Col
4. ces Nitrate to Nitrite 2 After a 20 minute incubation at room temperature Color Reagents A and B are added and incubated at room temperature for 5 minutes 3 The colored product is read and calculated as with the Nitrate determination above The concentration of Nitrate in the sample is calculated by subtracting the measured Nitrite concentration from the Total Nitric Oxide concentration for the sample This kit uses Nitrate and Nitrite Standard solutions calibrated to the US National Institute for Science and Technology Standard Reference Materials and ISO IEC standards B Bridge International Inc Nitric Oxide 041812 KIT COMPONENTS Clear 96 well plate Two Plates Nitrate Standard 2 000uM 200 uL Nitrite Standard 2 000uM 200 uL Assay Buffer 60 mL NADH Concentrate 1 2 mL Nitrate Reductase as a stable solid 1 vial Enzyme Stabilization Buffer 1 mL Color Reagent A 5 mL A solution of Sulfanilamide in acid CAUSTIC Color Reagent B 5 mL A solution of N 1 Naphthyl ethylenediamine in acid CAUSTIC Store above components at 4 C Once reconstituted the Nitrate Reductase must be stored at 20 MATERIALS REQUIRED BUT NOT SUPPLIED Distilled or deionized water free of detectable nitrate or nitrite 10 000 Molecular Weight Cut Off MWCO polysulfone filters Corning Spin X UF 500 431478 or similar product Repeater pipets using disposable tips for addition of Color Reagents A amp B NADH and Nitrate Reductase
5. d 4 200 ul Standard 5 200 ul Standard 6 200 ul Final Concentration 200 uM 100 uM 50 uM 25 uM 12 5 uM 6 25uM 3 125 uM B Bridge International Inc Nitric Oxide 041812 SAMPLE PREPARATION Nitrate Nitrite is identical across species and this kit will measure NO from sources other than human This kit will measure NO in cell culture medium however many media contain nitrate salts Care needs to be taken in the selection of media when NO measurement is to be done If samples need to be stored after collection we recommend storing them at 70 C or lower preferably after being frozen in liquid nitrogen This assay has been validated for serum plasma urine and saliva as well as water and buffer samples Tris HEPES and PBS buffers are compatible at pH 7 2 as is EDTA at lt 10 mM Detergents such as Triton X 100 Tween 20 and CHAPS are compatible at concentrations of lt 0 1 Most cell lysates and tissue homogenates should also be compatible Samples containing these detergents should be diluted at least 1 2 with the Assay Buffer Samples containing SDS or azide are not compatible with the assay Samples containing visible particulate should be centrifuged prior to filtration and using All samples must be filtered through a 10 000 MWCO spin filter to remove protein Serum plasma saliva or urine Dilute sample with Assay Buffer and filter through a 10 000 MWCO device following the manuf
6. f the regulatory damage facets of NO are major forces in mitochondrial signaling and dysfunction NO is linked not only to coronary heart disease endothelial dysfunctions erectile dysfunction and neurological disorders but also diabetes chronic periodontitis autism cancer and assorted age related diseases The physical properties of Nitric Oxide make it challenging for direct detection methods However colorimetric methods can be applied to measure its stable break down products nitrate NO3 and nitrite NOz ASSAY PRINCIPLE The B Bridge Nitric Oxide Detection Kit is designed to quantitatively measure Nitrate and Nitrite present in a variety of samples Nitric Oxide content is derived from the sum of Nitrate NO3 and Nitrite NOz Please read the complete kit insert before performing this assay Both Nitrate and Nitrite standards are provided to generate standard curves for the assay and all samples should be read off the appropriate standard curve For Nitrite detection 1 Samples are mixed with the Color Reagents A and B and incubated at room temperature for 5 minutes 2 The colored product is read at 550 570 nm 3 The concentration of Nitrite in the sample is calculated after making a suitable correction for any dilution of the sample using software available with most plate readers For total Nitric Oxide content 1 After the sample is incubated with Nitrate Reductase and NADH The reductase in combination with NADH redu
7. inutes For extended periods of time gt 2 hours store reconstituted NR on ice Store any unused reconstituted NR at 20 C Prepare NR for use in the assay by taking one part of reconstituted NR and adding to three parts of Assay Buffer See Table below Reconstituted NR 150 ul 275 ul ul Assay Buffer 450 ul 875 ul 1 5 mL Total Volume 600 ul 1 1 mL 2mL NADH Preparation Prepare NADH by diluting one part of NADH Concentrate with an equal part of Assay Buffer Do not store diluted NADH NADH Concentrate 300 ul 550 ul 1 mL Assay Buffer 300 ul 550 ul 1 mL Total Reaction Volume Mix 600 ul 1 1 mL 2 mL Standard Preparation Nitrate and Nitrite Standards are prepared identically by labeling seven test tubes as 1 through 7 Briefly vortex to mix and then spin the vial of standard in a microcentrifuge to ensure contents are at bottom of vial Pipet 360 uL of Assay Buffer into tube 1 and 200 uL into tubes 2 to 7 Carefully add 40 uL of either the NO or NO3 Standard to tube 1 and vortex completely Take 200 uL of the solution in tube 1 and add it to tube 2 and vortex completely Repeat this for tubes 3 through 7 The concentration of Nitrate or Nitrite in tubes 1 through 7 will be 200 100 50 25 12 5 6 25 and 3 125 uM Use all Standards within 2 hours of preparation Assay Buffer NOz or NO3 Standard 40 ul Standard 1 200 ul Standard 2 200 ul Standard 3 200 ul Standar
8. or Reagent B to each of well using a repeater pipet 8 Incubate at room temperature for 5 minutes 9 Read the optical density at 540 570 nm These readings are for the Total Nitric Oxide determination B Bridge International Inc Nitric Oxide 041812 CALCULATIONS Average the duplicate optical density readings for each standard and sample Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader after subtracting the mean ODs for the zero standard The concentrations obtained should be multiplied by the dilution factor to obtain sample values Nitrite NOz concentrations are calculated from the data obtained from the Nitrite Protocol standard curve data utilizing the curve fitting routine supplied with the plate reader Total NO concentrations are calculated from the data obtained from the Total Nitric Oxide Protocol nitrite nitrate standard curve data utilizing the curve fitting routine supplied with the plate reader Nitrate NO3 concentrations are obtained by subtracting the NO2 concentrations of each sample from the Total NO concentrations See Below Nitrate NO Total NO Nitrite NO TYPICAL DATA EXAMPLE ONLY TypicaL Data NITRITE Sample Mean OD Nitrite Conc uM Zero 0 038 O Standard 1 2 144 Standard 2 1 248 Standard 3 0 708 Standard 4 0 412 Standard 5 0 236 Standard 6 0 145 Standard 7 0 095 Sample 1 Sam
9. ple 2 Typical Data ToTAL Nitric OXIDE Sample Mean OD Total Nitric Oxide Conc uM Zero 0 040 O Standard 1 1 984 Standard 2 0 921 Standard 3 0 450 Standard 4 0 240 Standard 5 0138 Standard 6 0 092 Standard 7 Sample 1 Sample 2 B Bridge International Inc Nitric Oxide 041812 TYPICAL STANDARD CURVE EXAMPLE ONLY Standard curves vary with each assay Always run your own standard curves for calculation of results do not use this data 2 4 2 0 1 6 1 2 Net OD 0 8 0 4 0 0 Standard Conc pM Always run your own standard curves for calculation of results Do not use these data B Bridge International Inc Nitric Oxide 041812
10. uffer samples Tris HEPES and PBS buffers are compatible at pH 7 2 as is EDTA at lt 10 mM Detergents such as Triton X 100 Tween 20 and CHAPS are compatible at concentrations of lt 0 1 Most cell lysates and tissue homogenates should also be compatible Samples containing these detergents should be diluted at least 1 2 with the Assay Buffer Samples containing SDS or azide are not compatible with the assay Please read the complete kit insert before performing this assay This kit is species independent BACKGROUND Nitric oxide NO is a diffusible transient reactive molecule that has physiological effects in the picomolar to micromolar range Acting through soluble guanylate cyclase activation NO is an important physiological regulator of the cardiovascular nervous and immunological systems NO is bio available by two routes It can be endogenously generated by constitutive or induced enzymes like Nitric Oxide Synthase or it can be orally ingested as nitrates nitrites for rapid uptake into circulation and subsequent conversion The reactive nature of nitric oxide allows it to act as a cytotoxic factor when released during an immune response by cells such as macrophages The reactivity also allows NO to be easily converted to a toxic radical that can produce nitrosative damage to cells organelles and molecules such as DNA Nitrosaylation however can be a regulated post translational modification in cell signaling The balance and dynamics o
Download Pdf Manuals
Related Search