ab83362 Urea Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of Urea levels in various samples. This product is for research use only and is not intended for diagnostic use. Version 3 Last Updated 15 November 2013
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Table of Contents 1. Overview 3 2. Protocol Summary 4 3. Components and Storage 5 4. Assay Protocol 7 5. Data Analysis 9 6. Troubleshooting 10 2
1. Overview Urea is a waste product which produced in the liver, dissolved in blood (in a concentration of 2.5-7.5 mm), and secreted by the kidneys. Urea also plays a very important role in protein catabolism, removal of toxic ammonia from the body, and the counter-current system which allows for re-absorption of water and critical ions in the nephrons. Urea determination is very useful for the medical clinician to assess kidney and other organs function of patients. Abcam s Urea Assay Kit provides a rapid, simple, sensitive, and reliable for measurement of Urea level in a variety of samples such as serum, plasma, and urine, etc. In the assay, Urea reacts as substrate with compounds in the presence of enzymes to form a product that reacts with the OxiRed probe to generate color (λ max =570nm). The optical density of produced color has a direct relationship with Urea concentration in the solution. The kit can detect as low as 0.5 nmol per well or 10 µm of Urea. The assay is also suitable for high throughput studies. 3
2. Protocol Summary Sample Preparation Standard Curve Preparation A Add Reaction Mix Measure Optical Density 4
3. Components and Storage A. Kit Components Item Quantity Urea Assay Buffer 25 ml OxiRed Probe in DMSO 0.2 ml Enzyme Mix (Lyophilized) 1 vial Developer 1 vial Converting Enzyme 1 vial Urea Standard (100mM) 100 µl Store the kit at -20 C, protect from light. Allow Assay Buffer to warm to room temperature before use. Briefly centrifuge vials before opening. Read the entire protocol before performing the assay. All the solutions in this kit should be kept capped when not being used to prevent absorption of NH 3 from the air. UREA PROBE: Warm to room temperature before use. Store at -20 C, protect from light and moisture. 5
ENZYME MIX, DEVELOPER & CONVERTING ENZYME: Dissolve in 220 μl Assay Buffer separately. Aliquot to prevent frequent freeze/thaw cycles. Store at -20 C. Use within two months. B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker 6
4. Assay Protocol 1. Sample Preparation: a. For tissue or cell samples: Tissue (20 mg) or cells (2x10 6 ) can be homogenized in 100 μl Assay Buffer, centrifuge 15000 x g for 10 min to remove insoluble materials. 1-25 µl from the cell or tissue extracts can be added to the 96 well plates. Bring the volume to 50 µl/well with Assay buffer. b. For liquid samples: 1-25 μl samples (serum, plasma, urine, extracts or other liquid) can be directly added into 96 well plates. Bring the volume to 50 μl/well with Assay Buffer. For unknown samples, we suggest testing several doses of your sample to ensure the readings are within the standard curve range. Note: Ammonium ion, NAD + /NADP +, and pyruvate in the sample will interfere with the assay. These compounds can be eliminated by setting a sample control as described step 3. 2. Standard Curve Preparation: Dilute the Urea Standard to 0.5 mm by adding 5 μl of the 100 mm Urea Standard to 995 μl dh 2 O, mix well. Add 0, 2, 4, 6, 8, 10 μl into each well individually. Adjust volume to 50 μl/well with Assay Buffer to generate 0, 1, 2, 3, 4, 5 nmol/well of the Urea Standard. 7
3. Urea Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 μl Reaction Mix containing: Sample Sample Control Urea Assay Buffer 42 μl 44 μl OxiRed Probe 2 μl 2 μl Enzyme Mix 2 μl 2 μl Developer 2 μl 2 μl Converter Enzyme 2 μl --- Add 50 μl of the Reaction Mix to each well containing the Urea Standard and test samples. Add 50 μl of the Sample Control Mix to sample control well. Mix well. Incubate the reaction for 60 min at 37 C, protect from light. 4. Measure OD 570nm in a micro plate reader 8
5. Data Analysis Subtract zero nmol standard from all readings. Subtract sample control readings from sample readings. Plot Urea standard Curve. Apply the corrected sample readings to the standard curve. Urea concentrations of the test samples can then be calculated: Concentration = Sa / Sv (nmol/μl or mm) Where: Sa is the sample amount (nmol) of unknown Urea from standard curve. Sv is sample volume (μl) added into the wells. Urea Molecular Weight is 60.07g/mol. 9
6. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 10
Problem Reason Solution Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) 11
Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit For further technical questions please do not hesitate to contact us by email (technical@abcam.com) or phone (select contact us on www.abcam.com for the phone number for your region). 12
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