ab155901 Creatine Kinase Activity Assay Kit (Colorimetric) Instructions for Use For the sensitive and accurate measurement of Creatine Kinase activity in various samples. This product is for research use only and is not intended for diagnostic use.
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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 10 6. Troubleshooting 12 2
1. Overview Creatine Kinase (CK) also known as creatine phosphokinase (CPK) and ATP: creatine N-phosphotransferase is a common cellular enzyme (EC 2.7.3.2). It catalyzes the reversible conversion of creatine and ATP into ADP and phosphocreatine. CK is widely expressed in various tissues and cell types, with highest activity in striated muscles, heart tissue and brain. CK consists of two subunits: M (muscle) and B (brain), and has three isoenzymes: CK-MM (skeleton muscle), CK-MB (cardiac muscle), and CK-BB (brain). Increased CK level is associated with many diseases such as myocardial infarction, muscular dystrophy, pulmonary infarction and brain tumors. Accurate measurement of CK is crucial for early diagnosis, prediction and therapeutic strategy. In Abcam s Creatine Kinase Activity Colorimetric Assay kit, creatine kinase converts creatine into phosphocreatine and ADP. The generated phoshocreatine and ADP reacts with CK Enzyme Mix to form an intermediate, which reduces a colorless Probe to a colored product with strong absorbance at 450 nm. The CK Activity Assay is highthroughput adaptable, simple and sensitive. This assay kit can detect Creatine Kinase activity less than 1 mu. 3
2. Protocol Summary Reagent Preparation Standard Curve Preparation Sample Preparation Positive Control Reaction Mix Measurement and Calculation 4
3. Components and Storage A. Kit Components Item Quantity Assay Buffer 25 ml Substrate 1 ml ATP (Lyophilized) 1 vial Enzyme Mix (Lyophilized) 1 vial Developer (Lyophilized) 1 vial NADH Standard (Lyophilized) 1 vial Positive Control (Lyophilized) 1 vial * Store the kit at-20 C and protect from light. Please read the entire protocol before performing the assay. Avoid repeated freeze/thaw cycles. Warm all Buffers to room temperature before use. Briefly centrifuge all small vials prior to opening. 5
B. Additional Materials Required 96-well clear plate with flat bottoms Multi-well spectrophotometer (ELISA reader) 6
4. Assay Protocol A. Reagent Preparation 1. ATP: Reconstitute with 220 μl dh 2 O. Pipette up and down to dissolve completely. Aliquot and store at -20 C. Use within two months. 2. Enzyme Mix: Reconstitute with 220 μl Assay Buffer. Pipette up and down to dissolve completely. Aliquot and store at -20 C. Avoid repeated freeze/thaw cycles. Use within two months. Keep on ice while in use. 3. Developer: Reconstitute with 220 μl dh 2 O. Pipette up and down to dissolve completely. Aliquot and store at -20 C. Use within two months. Keep on ice while in use. 4. NADH Standard: Reconstitute with 50 μl Assay Buffer to generate 10.0 mm (10.0 nmol/μl) NADH Standard solution. Store at 20 C. Use within two months. Keep on ice while in use. 7
5. Positive control: Reconstitute with 200 μl CK Assay Buffer to generate 10 mu/μl stock & mix thoroughly. Aliquot & store at 20 C. Use within two months B. Creatine Kinase Assay Protocol 1. NADH Standard Curve: Dilute NADH Standard to 1 mm by adding 10 μl of 10 mm NADH Standard to 90 μl Assay Buffer. Add 0, 2, 4, 6, 8 and 10 μl of 1 mm NADH Standard into a series of wells in 96 well plate to generate 0, 2, 4, 6, 8 and 10 nmol/well of NADH Standard. Adjust volume to 50 μl/well with Assay Buffer. 2. Sample Preparation: Rapidly homogenize tissue (10 mg) or cells (1 x 10 6 ) with 100 μl ice cold Assay Buffer for 10 minutes on ice. Centrifuge at 12000 rpm for 5 min. Collect the supernatant. Add 1-50 μl sample 100µg) per well, adjust final volume to 50 μl with Assay Buffer. For Positive Control, add 2-10 μl of Positive Control into desired well(s). Adjust final volume to 50 μl with Assay Buffer. Note: Small molecules such as ADP, NADH etc. in some tissue samples such as liver may generate background. To remove small molecules, we suggest using 10K spin column 8
For unknown samples, we suggest testing several doses to ensure the readings are within the Standard Curve range. 3. Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare 50 μl Reaction Mix containing: Reaction Mix Assay Buffer 34 µl Enzyme Mix 2 µl Developer 2 µl ATP 2 µl Substrate 10 µl Add 50 μl of the Reaction Mix to each well containing the Standard, Positive Control and samples, mix well. 4. Measurement: Incubate for 20-40 min at 37 o C and measure OD 450nm. Note: Incubation time depends on the Creatine Kinase activity in the samples. We recommend measuring the OD in a kinetic mode and choose two time points (T1 & T2) in the linear range to calculate the CK activity of the samples. The NADH Standard 9
curve can read in Endpoint mode (ie: at the end of incubation time). 5. Data Analysis Calculation: Subtract 0 Standard reading from all readings. Plot the NADH Standard Curve. Calculate the Creatine Kinase activity of the test sample: ΔOD = A2 A1. Apply the ΔOD to the NADH Standard Curve to get B nmol of NADH generated by Creatine Kinase during the reaction time (ΔT = T2 - T1). Sample Creatine Kinase Activity = B T X V x Dilution Factor = nmol/min/ml/ = mu/ml Where: B is the NADH amount from the standard curve (nmol) T is the reaction time (min) V is the sample volume added into the reaction well (ml). Unit Definition: One unit of Creatine Kinase is the amount of enzyme that will generate 1.0 μmol of NADH per min at ph 9.0 at 37 0 C. 10
Figure 1: NADH Standard curve (a). Creatine Kinase activity in human serum (5 μl) & rat heart lysate (192 ng) (b). Assays were performed following kit protocol. 11
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 12
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) 13
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). 14
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