ab65340 Creatinine Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of Creatinine in various samples. This product is for research use only and is not intended for diagnostic use. Version: 2 Last Updated: 23 August 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 11 2
1. Overview Creatinine is a breakdown product of creatine phosphate. Creatinine is produced and excreted at a constant rate, and blood creatinine is used to determine glomerular filtration rate (GFR), a measure of kidney function. Blood creatinine levels increase only in cases of significant (>75%) damage to nephrons. Creatinine clearance is frequently used as a means of standardizing excretion of other compounds such as isoprostanes. Abcam's Creatinine Assay Kit provides an accurate, convenient measure of creatine concentration in biological fluids such as serum, urine or CSF. In the assay, creatinine is converted to creatine by creatininase, creatine is converted to sarcosine, which is specifically oxidized to produce a product which reacts with a probe to generate red color (λ max = 570 nm) and fluorescence (Ex/Em = 538/587 nm). Unlike picric acid assays, this kit is suitable for serum/plasma creatinine determinations, as well as for urine and other biological samples. 3
2. Protocol Summary Standard Preparation Sample Preparation Add Reaction Mix Measure Optical Density or Fluorescence 4
3. Components and Storage A. Kit Components Item Quantity Creatinine Assay Buffer 25 ml Creatinine Probe (in DMSO) 200 μl Creatinase (Lyophilized) 1 vial Creatininase (Lyophilized) 1 vial Creatinine Enzyme Mix (Lyophilized) 1 vial 10 µmol Creatinine (Lyophilized) 1 vial * Store kit at -20 C CREATININE ASSAY BUFFER: Ready to use as supplied. It may be stored at 4 C or -20 C CREATININE PROBE: Ready to use as supplied. Warm to room temperature to melt frozen DMSO prior to use. Store at -20 C, protect from light and moisture. Stable for at least 2 months. CREATININASE, CREATINASE AND CREATININE ENZYME MIX: Reconstitute with 220 μl of Assay Buffer. Keep on ice during use. 5
Store at -20 C when not in use. Aliquot each and store until needed. Freeze/thaw should be limited to one time. CREATININE STANDARD: Reconstitute with 100 μl of dh 2 O to generate 100 mm Creatinine Standard. Dissolve completely. Store at -20 C, stable for 2 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. Standard Preparation: a. For the colorimetric assay: Mix 10 μl of Creatinine Standard with 990 μl of Assay Buffer to generate 1 nmol/μl standard working solution. Add 0, 2, 4, 6, 8, 10 μl of the working solution to 6 consecutive wells. Bring the volume of each to 50 μl with Assay Buffer. b. For the fluorometric assay: If a more sensitive assay is desired, fluorescence can be utilized. Dilute the standard working solution 10-100 fold, and follow the same procedure as for the colorimetric assay. Slightly better results are obtained with the fluorescent assay by diluting the probe 10X with DMSO. 2. Sample Preparation: High concentrations of protein may interfere with the assay. Samples containing protein may be filtered through a 10kDa MW cut-off filter (ab93349) prior to assay. Add 2-50 μl of sample to the wells and bring the volume to 50 μl with Assay buffer. Note: Serum contains ~45-110 pmol/μl of creatinine. For unknown samples, we suggest testing several different dilutions to ensure the readings are in the linear range of the standard curve. background control should be performed by replacing 2 µl 7
Creatininase with 2 µl Creatinine Assay Buffer. The background should be subtracted from all readings. Endogenous compounds in the sample may interfere with the assay, so it is suggested to spike samples with a known amount of Creatinine (0-10 nmol) to ensure accurate determinations of creatinine in your sample. 3. Prepare Reaction Mix: Prepare enough reaction mix for the standard and samples. For each assay use: Samples Background Assay Buffer 42 μl 44 μl Creatinase 2 μl 2 μl Creatininase* 2 μl --- Enzyme Mix 2 μl 2 μl Probe** 2 μl 2 μl Mix well. Add 50 μl of the appropriate Reaction Mix to each standard and sample well, mix. Incubate at 37 C for 1 hr. * Note: Sarcosine and creatine generate background. If significant amounts of sarcosine or creatine are present in your samples, they can be measured by preparing a reaction without the creatininase (replace the 2 μl creatininase with 2 μl assay buffer) then the background can be subtracted from creatinine readings. 8
** Note: For the fluorescence assay, if the fluorescence background is too high, 0.4 μl of the probe can be use for each stands and samples, which will decrease the background reading significantly. 4. Read the plate in a plate reader at 570 nm, or fluorescence with Ex/Em = 538/587 nm. 5. Data Analysis 1. Plot standard curve: Subtract reagent background from all the readings. Plot readings vs. nmoles creatinine. 2. Determine sample Creatinine concentrations: Subtract sarcosine and creatine background from creatinine samples. Apply the creatinine reading to the standard curve. Concentration = Sa / Sv nmol/μl, or mm Where: Sa is the sample amount of unknown in nmol from your standard curve. Sv is the sample volume added to the well in micro-liter. Creatinine molecular weight: 113.12. 9
Creatinine Assay performed according to instructions. Fluorescent assay utilized 10X dilution of probe to reduce background 10
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 11
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) 12
Problem Reason Solution 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). 13
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