Epigenase™ 5-mC Hydroxylase TET Activity/Inhibition
Contents
1. Well Strip 1 Sirip 2 Strip 3 Strip 4 Strip5 Strip 6 A Blank Blank Sample Sample Sample Sample B TAS 0 05 ng ul TAS 0 05 ng ul Sample Sample Sample Sample Cc TAS 0 1 ng ul TAS 0 1 ng ul Sample Sample Sample Sample D TAS 0 2 ng ul TAS 0 2 ng ul Sample Sample Sample Sample E TAS 0 5 ng ul TAS 0 5 ng ul Sample Sample Sample Sample F TAS 1 ng ul TAS 1 ng ul Sample Sample Sample Sample G Internal control Internal control Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 9 Printed 2014 09 22 P 3094 TROUBLESHOOTING Problem Possible Cause Suggestion No signal or weak signal in both the standard and sample wells Reagents are added incorrectly Check if reagents are added in the proper order with the right amount and if any steps in the protocol may have been omitted by mistake The substrate and standard are not properly bound to the wells Ensure that 1 the TS and TAS are added into the wells 2 the wells are completely covered with sufficient BS Binding Solution and 3 binding time is sufficient 90 min Incubation time and temperature are incorrect Ensure the incubation time and temperature2. Epigenase Thymine DNA Glycosylase Activity Inhibition Assay Kit Colorimetric Base Catalog P 3094 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The Epigenase Thymine DNA Glycosylase Activity Inhibition Assay Kit Colorimetric is suitable for measuring the activity inhibition of total TDG enzyme using nuclear extracts or purified TDG enzymes from a broad range of species such as mammals plants fungi and bacteria ina variety of forms including but not limited to cultured cells and fresh and frozen tissues Starting Materials Input materials can be nuclear extracts or purified TDG enzymes The amount of nuclear extracts for each assay can be between 2 yg to 10 ug with an optimal range of 5 6 ug The amount of purified enzymes can be between 20 ng to 1 ug depending on the purity and catalytic activity of the enzymes Internal Control The TDG assay standard is provided in this kit for the quantification of TDG enzyme activity Because TDG activity can vary from tissue to tissue and from normal and diseased states it is advised to run replicate samples to ensure that the signal generated is validated Precautions To avoid cross contamination carefully pipette the sample or solution into the strip wells Use aerosol barrier pipette tips and always change pipette tips between liquid transfers Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi Cou
3. and development in the wells consistent with the order you added the other reagents e g from well A to G or from well 1 to 12 RELATED PRODUCTS Nuclear Extract Preparation OP 0002 1 EpiQuik Nuclear Extraction Kit DNA Methylation P 1015 Methylamp Methylated DNA Capture Kit P 2019 EpiQuik Methylated DNA Immunoprecipitation Kit P 2020 EpiQuik Tissue Methylated DNA Immunoprecipitation Kit P 1034 MethylFlash Methylated DNA Quantification Kit Colorimetric P 1035 MethylFlash Methylated DNA Quantification Kit Fluorometric P 1039 MethylFlash Urine 5 Methylcytosine Quantification Kit Colorimetric P 1040 MethylFlash Urine 5 Methylcytosine Quantification Kit Fluorometric P 3009 EpiQuik DNMT Activity Inhibition Assay Ultra Kit Colorimetric P 3010 EpiQuik DNMT Activity Inhibition Assay Ultra Kit Fluorometric DNA Demethylation P 1036 MethylFlash Hydroxymethylated DNA Quantification Kit Colorimetric P 1037 MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric P 1038 EpiQuik Hydroxymethylated DNA Immunoprecipitation nMeDIP Kit P 3087 Epigenase 5mC Hydroxylase TET Activity Inhibition Kit Fluorometric P 1041 MethylFlash 5 Formylcytosine 5 fC DNA Quantification Kit 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Produ
4. described in the protocol are followed correctly Incorrect absorbance reading Check if the appropriate absorbance wavelength 450 nm filter is used Kit was not stored or handled properly Ensure all components of the kit are stored at the appropriate temperatures and tightly capped after each opening or use No signal or weak signal in only the standard curve wells The standard amount is insufficiently added to the well in Step 2c Ensure a sufficient amount of standard is added The standard is degraded due to improper storage conditions Follow the Shipping amp Storage guidance of this User Guide for storage of TAS TDG Assay Standard High background present in the blank wells Insufficient washing of wells Check if washing at each step is performed according to the protocol Contaminated by sample or standard Ensure the well is not contaminated from adding sample or standard accidentally or from using contaminated tips Incubation time with detection antibody is too long The incubation time at Step 3d should not exceed 45 minutes Over development of color Decrease the development time in Step 4a before adding SS Stop Solution in Step 4b No signal or weak signal only in sample wells Protein sample is not properly extracted or purified Ensure your protocol is suitable for TDG protein extraction For the best results it is advised to use Ep
5. determining cellular response to chemotherapy through DNA demethylation and BER thereby benefiting cancer diagnostics and new target based cancer therapeutics The currently used methods for TDG activity detection involve DNA cleavage followed by electrophoresis which can be time consuming and inconvenient with low throughput and high cost To address this issue Epigentek developed and offers the Epigenase Thymine DNA Glycosylase TDG Activity Inhibition Assay Kit Colorimetric e Colorimetric assay with easy to follow steps for convenience and speed The entire procedure can be finished within 5 hours e Directly measures TDG activity without the need for DNA cleavage electrophoresis or chromatography e Both cell tissue extracts and purified TDG proteins can be used which allows for detection of inhibitory effects of TDG inhibitors in vivo and in vitro e Novel assay principle allows high sensitivity to be achieved The activity inhibition of TDG can be detected from as little as 20 ng of purified TDG proteins e TDG assay standard is conveniently included which allows for specific quantification of TDG activity e Strip microplate format makes the assay flexible manual or high throughput analysis 96 assays PRINCIPLE amp PROCEDURE In this assay a 5 fC DNA substrate is stably coated onto microplate wells Active TDG binds to the substrate and excises 5 fC from the substrate by a nicking reaction The remaining un excised 5 fC
6. without inhibitor Total volume should be 50 ul per well Sample Wells With Inhibitor Add 41 to 44 ul of TAB 1 to 4 ul of nuclear extracts or purified TDG enzyme and 5 ul of inhibitor solution Total volume should be 50 ul per well Note 1 Follow the suggested well setup diagrams under Suggested Strip Well Setup 2 It is recommended to use 5 ug to 10 ug of nuclear extract per well or 50 ng to 500 ng of purified enzyme per well 3 The concentration of inhibitor to be added into the sample wells can be varied 1 uM to 1000 uM However the final concentration of the inhibitors before adding to the wells should be prepared with TAB at a 1 10 ratio i e add 0 5 ul of inhibitor to 4 5 ul of TAB so that the original solvent of the inhibitor can be reduced to 1 of the reaction solution or less Tightly cover strip plate with Parafilm M to avoid evaporation and incubate at 37 C for 45 60 min Note 1 The incubation time may depend on intrinsic TDG activity However in general 45 min incubation is suitable for active purified TDG enzyme and 60 min incubation is required for nuclear extract Remove the reaction solution from each well Wash each well three times with 150 ul of the Diluted WB 1X Wash Buffer each time 3 Antibody Binding amp Signal Enhancing a Add 50 ul of the Diluted CA to each well then cover with Parafilm M or aluminium foil and incubate at room temperature for 60 min b Remove the Diluted CA solution f
7. at disposable gloves and proper eye protection are required when working with this product Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design The information in this User Guide is subject to change at any time without notice Therefore only use the User Guide that was supplied with the kit when using that kit Usage Limitation The Epigenase Thymine DNA Glycosylase TDG Activity Inhibition Assay Kit Colorimetric is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The Epigenase Thymine DNA Glycosylase TDG Activity Inhibition Assay Kit Colorimetric and methods of use are intellectual property of Epigentek A BRIEF OVERVIEW DNA methylation occurs by the covalent addition of a methyl group at the 5 carbon of the cytosine ring by DNA methyltransferases resulting in 5 methylcytosine 5mC In somatic cells 5mC is found almost exclusively in the context of paired symmetrical methylation of the dinucleotide CpG whereas in embryonic stem ES cells a substantial amount of 5mC is also observed in non CpG contexts The biological importance of 5mC as amajor epigenetic modifier of phenotype and gene expression has been widely recognized DNA demethylation has been demonstrated to occur through at least two different pathways 1 active deamination of 5 mC to T by an AID APOBEC enzyme giving a G T mismatch that is
8. bag tightly and store at 4 C b Add 80 ul of BS Binding Solution to each well c Add 2 ul of TAB into the blank wells Add 2 ul of 0 5X TS into internal control wells and each sample well Add 1 ul of Diluted TAS into the standard curve wells see the designated wells depicted in Table 1 under Suggested Strip Well Setup below Mix solution by gently tilting from side to side or shaking the plate several times Ensure the solution coats the bottom of the well evenly Note For the standard curve add 1 ul of Diluted TAS at concentrations of 0 05 to 1 ng ul see the chart in Step 19 The final concentrations should be 0 05 0 1 0 2 0 5 and 1 ng per well d Cover strip plate with plate seal or Parafilm M and incubate at 37 C for 90 min 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 22 P 3094 Remove the BS Binding Solution from each well Wash each well three times with 150 ul of the Diluted WB 1X Wash Buffer each time Blank Wells Add 50 ul of TAB to each blank well Standard Wells Add 50 ul of TAB to each standard well Internal Control Wells Add 50 ul of TAB to each internal control well Sample Wells Without Inhibitor Add 46 to 49 ul of TAB and 1 to 4 ul of nuclear extracts or purified TDG enzyme to each sample well
9. ch assay well Prepare Diluted DA Detection Antibody Solution Dilute DA Detection Antibody with Diluted WB 1X Wash Buffer at a ratio of 1 2000 i e add 1 ul of DA to 2000 ul of Diluted WB 1X Wash Buffer About 50 ul of Diluted DA will be required for each assay well Prepare Diluted ES Enhancer Solution Dilute ES Enhancer Solution with Diluted WB 1X Wash Buffer at a ratio of 1 5000 i e add 1 ul of ES to 5000 ul of WB 1X Wash Buffer About 50 ul of Diluted ES will be required for each assay well Prepare Diluted TAS Standard Solution Suggested Standard Curve Preparation First dilute TAS with TAB to 1 ng ul by adding 1 ul of TAS to 9 ul of TAB Then further prepare five concentrations by combining the 1 ng ul Diluted TAS with TAB into final concentrations of 0 05 0 1 0 2 0 5 and 1 0 ng ul according to the following dilution chart Resulting TA TAS 1 now TaB Besuting TAS 1 1 0 ul 19 0 ul 0 05 ng ul 2 1 0 ul 9 0 ul 0 1 ng ul 3 1 0 ul 4 0 ul 0 2 ng ul 4 2 0 pl 2 0 ul 0 5 ng ul 5 4 0 ul 0 0 ul 1 ng ul Note Keep each of the diluted solutions except Diluted WB 1X Wash Buffer on ice until use Any remaining diluted solutions other than Diluted WB should be discarded if not used within the same day 2 Enzymatic Reaction a Predetermine the number of strip wells required for your experiment Carefully remove un needed strip wells from the plate frame and place them back in the bag seal the
10. converted to G C by thymine DNA glycosylase TDG and then subsequently base excision repair BER and 2 iterative oxidation of 5 mC by TET enzyme to 5 hmC and further to 5 formycytosine 5 fC and 5 carboxylcytosine 5 caC Both 5 fC and 5 caC can be rapidly excised by TDG to allow subsequent BER processing which results in 5 fC and 5 caC converting back to unmodified cytosine 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 22 P 3094 mc hmc fC cac cC Ao Oxidation NH ER NH a LAO E koo 106 ae no Iterative LA Base excisi excision Ay 1 oxidation i repair wan DNA X CHO or COO NA TDG belongs to the TDG mug DNA glycosylase family Besides playing a critical role in active DNA methylation TDG also removes thymine moieties from G T mismatches by hydrolyzing the carbon nitrogen bond between the sugar phosphate backbone of DNA and the mismatched thymine It has been shown that TDG participates in the regulation of embryonic and germ cell development mediates cellular defense against genetic and epigenetic mutations and mediates DNA directed cytotoxicity In addition TDG is a proposed tumor suppressor candidate Thus the measurement of TDG activity would be important as TDG may be involved in preventing tumorigenesis and
11. cts are for research use only Page 11 Printed 2014 09 22 P 3094
12. igentek s Nuclear Extraction Kit Cat No OP 0002 Also use fresh cells or tissues for protein extraction as frozen cells or tissues could lose enzyme activity Sample amount added into the wells is insufficient Ensure a sufficient amount of purified enzymes or nuclear extracts is used as 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 10 Printed 2014 09 22 P 3094 indicated in Steps 2J and 2K The sample can be titrated to determine the optimal amount to use in the assay Sample was not stored properly Ensure sample is stored in aliquots at or has been stored for too long 80 C for no more than 6 weeks for nuclear extracts and 6 months for purified enzymes Avoid repeated freezing thawing Little or no activity of TDG This problem may be a result of many contained in the sample factors If the affecting factors cannot be determined use new or re prepared nuclear extracts or purified enzymes Uneven color development Insufficient wash of the wells Ensure the wells are washed according to the guidance of washing and residual wash buffer is removed as much as possible Delayed color development or Ensure color development and stop delayed stopping of color solutions are added sequentially
13. in the substrate will be recognized with a high affinity 5 fC DNA antibody The ratio or amount of 5 fC in the substrate which is inversely proportional to enzyme activity can then be colorimetrically measured by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm The activity of the TDG enzyme is in turn inversely proportional to the optical density intensity measured 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3094 0 6 R 0 89915 0D450 nm 0 0 2 04 Os 08 1 TDG Assay Standard ng Fig 1 Illustrated standard curve generated with the TDG assay standard from the Epigenase Thymine DNA Glycosylase TDG Activity Inhibition Assay Colorimetric ASSAY PROTOCOL os amp gt Total TOG Activity OD hour gt o v 01 o 2 5 10 Nuclear extracts ug Fig 2 Demonstration of high sensitivity and specificity of the TDG activity assay achieved by using nuclear extracts with the the Epigenase Thymine DNA For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input Amount The amount of nuclear extracts for each assay can be between 2 ug and 10 yg with an optimal range of 5 6 ug The amount of pu
14. k is 0 1 Protein amount is 5 pg Incubation time is 1 hour 60 min 0 65 0 35 TDG activity _ S gt X 1000 1 OD min mg 5 xX 60 Page 8 Printed 2014 09 22 P 3094 For accurate or specific activity calculation First generate a standard curve and plot the OD values versus the amount of TAS at each concentration point Then determine the slope as OD ng using linear regression Microsoft Excel s linear regression or slope functions are suitable for such calculations and the most linear part include at least 4 concentration points of the standard curve for optimal slope calculation Now calculate the amount of excised 5 fC DNA using the following formulas Internal Control OD blank OD Sample OD Blank OD Excised 5 fC DNA ng Slope Excised Substrate ng TDG Activity ng min mg x 1000 Protein Amount ug X min Protein amount added into the reaction at step 2J Incubation time at step 2L in minutes For inhibition calculation Internal Control OD blank OD Inhibitor Sample OD Blank OD Inhibition 1 M Internal Control OD blank OD No Inhibitor Sample OD Blank OD X 100 SUGGESTED STRIP WELL SETUP Table 1 The suggested strip well plate setup for standard curve preparation in a 48 assay format in a 96 assay format Strips 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate
15. nty Blvd Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3094 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 3094 48 Cat P 3094 96 Upon Receipt WB 10X Wash Buffer 14 ml 28 ml 47 TAB TDG Assay Buffer 3 ml 6 ml RT TS 10X TDG Substrate 10 pl 20 ul 20 C BS Binding Solution 5 ml 10 ml RT TAS TDG Assay Standard 10 ug ml 10 ul 20 ul 20 C CA Capture Antibody 1000X 4ul 8 ul 4 DA Detection Antibody 2000X 5 ul 10 ul 20 C ES Enhancer Solution 5 ul 10 ul 20 C DS Developer Solution 5 ml 10 ml 47 SS Stop Solution 5 ml 10 ml RT 8 Well Assay Strips With Frame 6 12 47 User Guide 1 1 RT For maximum recovery of the products centrifuge the original vial prior to opening the cap SHIPPING amp STORAGE The kit is shipped in three parts the first part at ambient room temperature and the second and third parts on frozen ice packs at 4 C Upon receipt 1 Store TS TAS DA and ES at 20 C away from light 2 Store WB CA DS and 8 Well Assay Strips at 4 C away from light 3 Store remaining components TAB BS and SS at room temperature away from light Note Check if WB 10X Wash Buffer contains salt precipitates before use If so warm at ro
16. om temperature or 37 C and shake the buffer until the salts are re dissolved All components of the kit are stable for 6 months from the date of shipment when stored properly MATERIALS REQUIRED BUT NOT SUPPLIED oO Oo O OF 0 0 o oO Adjustable pipette or multiple channel pipette Multiple channel pipette reservoirs Aerosol resistant pipette tips Microplate reader capable of reading absorbance at 450 nm 1 5 ml microcentrifuge tubes Incubator for 37 C incubation Distilled water Nuclear extract or purified enzymes 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 2 Printed 2014 09 22 P 3094 O Parafilm M or aluminum foil GENERAL PRODUCT INFORMATION Quality Control Each lot of Epigenase Thymine DNA Glycosylase TDG Activity Inhibition Assay Kit Colorimetric is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described in our product instructions Product Warranty If this product does not meet your expectations simply contact our technical support unit or your regional distributor We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques Safety Suitable lab co
17. r will change to yellow after adding SS and the absorbance should be read ona microplate reader within 2 to 10 min at 450 nm with an optional reference wavelength of 655 nm Note 1 Most microplate readers have the capability to carry out dual wavelength analysis and will automatically subtract reference wavelength absorbance from the test wavelength absorbance If your plate reader does not have this capability the plate can be read twice once at 450 nm and once at 655 nm Then manually subtract the 655 nm ODs from 450 nm ODs 2 If the stripwell microplate frame does not fit in the microplate reader transfer the solution to a standard 96 well microplate 5 TDG Activity Calculation 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Calculate the average duplicate readings for sample wells and blank wells Calculate TDG activity or inhibition using the following formulas For simple calculation Internal Control OD blank OD Sample OD Blank OD TDG Activity OD min mg _ x 1000 Protein Amount ug X min Protein amount added into the reaction at step 2J Incubation time at step 2L in minutes Example calculation Average OD450 of internal control is 0 75 Average OD450 of sample is 0 45 Average OD450 of blan
18. rified enzymes can be between 20 ng and 1 ug depending on the purity and catalytic activity of the enzymes Nuclear Extraction You can use your method of choice for preparing nuclear extracts Epigentek offers a nuclear extraction kit Cat OP 0002 optimized for use with this kit Nuclear Extract or Purified TDG Protein Storage Nuclear extract or purified TDG enzyme should be stored in aliquots at 80 C until use 1 Buffer Solution amp Preparation a Prepare Diluted WB 1X Wash Buffer 48 Assay Kit Add 13 ml of WB 10X Wash Buffer to 117 ml of distilled water and adjust pH to 7 2 7 5 96 Assay Kit Add 26 ml of WB 10X Wash Buffer to 234 ml of distilled water and adjust pH to 7 2 7 5 This Diluted WB 1X Wash Buffer can now be stored at 4 C for up to six months b Prepare 0 5X TS TDG Substrate Add 1 ul of TS 10X TDG Substrate to 19 ul of TAB Assay Buffer About 2 ul of 0 5X TS will be required for each assay well 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3094 Prepare Diluted CA Capture Antibody Solution Dilute CA Capture Antibody with Diluted WB 1X Wash Buffer at a ratio of 1 1000 i e add 1 ul of CA to 1000 ul of Diluted WB 1X Wash Buffer About 50 ul of Diluted CA will be required for ea
19. rom each well c Wash each well three times with 150 ul of the Diluted WB 1X Wash Buffer each time d Add 50 ul of the Diluted DA to each well then cover with Parafilm M or aluminium foil and incubate at room temperature for 30 min e Remove the Diluted DA solution from each well f Wash each well four times with 150 ul of the Diluted WB 1X Wash Buffer each time g Add 50 ul of the Diluted ES to each well then cover with Parafilm M or aluminum foil and incubate at room temperature for 30 min h Remove the Diluted ES solution from each well 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 7 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3094 Wash each well five times with 150 ul of the Diluted WB 1X Wash Buffer each time Note Ensure any residual wash buffer in the wells is thoroughly removed at each wash step The wash can be carried out by simply pipetting the wash buffer into the wells and then pipetting the buffer out from the wells discard the buffer 4 Signal Detection a Add 100 ul of DS to each well and incubate at room temperature for 1 to 10 min away from light Begin monitoring color changes in the sample wells and control wells Add 50 ul of SS to each well to stop enzyme reaction when the color in the positive control wells turns medium blue The colo
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