Epigenase™ 5mC Hydroxylase TET Activity/Inhibition
Contents
1. Epigenase 5mC Hydroxylase TET Activity Inhibition Assay Kit Colorimetric Base Catalog P 3086 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The Epigenase 5mC Hydroxylase TET Activity Inhibition Assay Kit is suitable for measuring the activity inhibition of total 5nC hydroxylase TET enzyme using nuclear extracts or purified TET isoforms TET 1 3 from a broad range of species such as mammalian plant fungal and bacterial in a variety of forms including but not limited to cultured cells fresh and frozen tissues Starting Materials Input materials can be nuclear extracts or purified TET enzymes The amount of nuclear extracts for each assay can be 2 yg to 20 ug with an optimal range of 5 10 ug The amount of purified enzymes can be 20 ng to 1 ug depending on the purity and catalytic activity of the enzymes Internal Control The TET assay standard 5 hydroxymethylcytosine is provided in this kit for the quantification of TET enzyme activity Because TET 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 immedia2. 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 TET protein extraction For the best results it is advised to use Epigentek s Nuclear Extraction Kit Cat No OP 0002 Also use fresh 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 3086 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 indicated in Steps 2i and 2j The sample can be titrated to determine the optimal amount to use in the assay Sample was not stored properly or has been stored for too long Ensure sample is stored in aliquots at 80 C with no more than 6 weeks for nuclear extracts and 6 months for purified enzymes Avoid repeated freezing thawing Little or no activity of TET contained in the sample This problem may be a result of many factors If the affecting factors cannot be determined use new or re prepared nucle
3. standard are not properly bound to the wells Ensure that 1 the TS and HC5 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 temperature 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 were stored at the appropriate temperatures and the cap is 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 HC5 TET 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
4. 1 0 ul 9 0 ul 0 2 ng ul 3 1 0 ul 3 0 ul 0 5 ng ul 4 2 0 ul 2 0 ul 1 0 ng ul 5 4 0 ul 0 0 ul 2 0 ng pl Note Keep each of 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 bag tightly and store at 4 C Add 80 ul of BS Binding Solution to each well Add 2 ul of 0 5X TS into each blank well and each sample well Add 1 ul of Diluted HC5 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 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 3086 Note For the standard curve add 1 ul of Diluted HC5 at concentrations of 0 05 to 2 ng ul see the chart in Step 19 The final concentrations should be 0 05 0 2 0 5 1 and 2 ng per well Cover strip plate with plate seal or Parafilm M and incuba
5. 9 22 Epigentek Group Inc All rights reserved Products are for research use only P 3086 Prepare 0 5X TS TET Substrate Add 1 ul of TS 10X TET Substrate to 19 ul of TAB Assay Buffer About 2 ul of 0 5X TS will be required for each assay well Prepare Diluted HC6 Capture Antibody Solution Dilute HC6 Capture Antibody with Diluted WB 1X Wash Buffer at a ratio of 1 1000 i e add 1 ul of HC6 to 1000 ul of Diluted WB 1X Wash Buffer About 50 ul of Diluted HC6 will be required for each assay well Prepare Diluted HC7 Detection Antibody Solution Dilute HC7 Detection Antibody with Diluted WB 1X Wash Buffer at a ratio of 1 2000 i e add 1 ul of HC7 to 2000 ul of Diluted WB 1X Wash Buffer About 50 ul of Diluted HC7 will be required for each assay well Prepare Diluted HC8 Enhancer Solution Dilute HC8 Enhancer Solution with Diluted WB 1X Wash Buffer at a ratio of 1 5000 i e add 1 ul of HC8 to 5000 ul of WB 1X Wash Buffer About 50 ul of Diluted HC8 will be required for each assay well Prepare Diluted HC5 Standard Solution Suggested Standard Curve Preparation First dilute HC5 with TAB to 2 ng ul by adding 1 ul of HC5 to 9 ul of TAB Then further prepare five concentrations by combining the 2 ng l Diluted HC5 with TAB into final concentrations of 0 05 0 2 0 5 1 0 and 2 0 ng ul according to the following dilution chart Resulting HC5 Concentration Tube HC5 2 ng pl TAB 1 1 0ul 39 0ul 0 05 ng ul 2
6. ASSAY PROTOCOL 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 2 ug to 20 yg with an optimal range of 5 10 ug The amount of purified enzymes can be 20 ng to 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 TET Protein Storage Nuclear extract or purified TET 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 Final TAB TET Assay Buffer Add Co Factor 1 Co Factor 2 and Co Factor 3 to TAB Assay Buffer at a ratio of 1 100 i e add 1 ul of each Co Factor to 100 ul of TAB for a total of 103 pl About 50 ul of Final TAB 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 0
7. OD Inhibition 1 X 100 No Inhibitor Sample OD Blank OD 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 Well Strip 1 Strip 2 Strip3 Strip4 Strip 5 Strip 6 A Blank Blank Sample Sample Sample Sample B HC5 0 05 ng ul HC5 0 05 ng ul Sample Sample Sample Sample Cc HC5 0 2 ng ul HC5 0 2 ng ul Sample Sample Sample Sample D HC5 0 5 ng ul HC5 0 5 ng ul Sample Sample Sample Sample E HC5 1 ng ul HC5 1 ng ul Sample Sample Sample Sample F HC5 2 ng ul HC5 2 ng ul Sample Sample Sample Sample G Sample Sample 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 Printed 2014 09 22 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
8. Protein amount added into the reaction at step 2i Incubation time at step 2k in minutes Example calculation Average OD450 of sample is 0 65 Average OD450 of blank is 0 05 Protein amount is 5 pg Incubation time is 1 hour 60 min 0 65 0 05 TET activity X 1000 2 OD min mg 5 xX 60 For accurate or specific activity calculation 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 8 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 3086 First generate a standard curve and plot the OD values versus the amount of HC5 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 calculation and the most linear part include at least 4 concentration points of the standard curve for optimal slope calculation Now calculate the amount of HC5 converted hydroxymethylated product using the following formulas Sample OD Blank OD Hydroxymethylated product ng Slope Hydroxymethylated Product ng TET Activity ng min mg x 1000 Protein Amount ug X min Protein amount added into the reaction at step 2i Incubation time at step 2k in minutes For inhibition calculation Inhibitor Sample OD Blank
9. ar extracts or purified enzymes Uneven color Insufficient wash of the wells development Ensure the wells are washed according to the guidance of washing and residue washing buffer is removed as much as possible Delayed color development or delayed stopping of color development in the wells Ensure color development and stop solutions are added sequentially and 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 Hydroxymethylation 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 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 11 Printed 2014 09 22 P 3086
10. d TET enzyme and 90 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 HC6 to each well then cover Parafilm M or aluminium foil and incubate at room temperature for 60 min b Remove the Diluted HC6 solution from 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 HC7 to each well then cover with Parafilm M or aluminium foil and incubate at room temperature for 30 min e Remove the Diluted HC7 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 HC8 to each well then cover with Parafilm M or aluminum foil and incubate at room temperature for 30 min 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 3086 Remove the Diluted HC8 solution from each well 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 was
11. ethylcytosine 5hmC as a sixth DNA base with functions in transcription regulation has been detected to be abundant in human and mouse brain and embryonic stem ES cells In mammals it can be generated by oxidation of 5mC a reaction mediated by the ten eleven translocation TET family of 5mC hydroxylases 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 Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3086 HH H H H Hae N H CH CHOH N DNMTs N 3 TETs N 2 t AS A o 0 N o c sme hmg Unmethylated DNA Methylated DNA Hydroxymethylated DNA T C G T C G A C G T C G T C G A C G T C G T C G A C G The TET family of 5mC hydroxylases includes TET1 TET2 and TET3 These TET proteins may promote DNA demethylation by binding to CpG rich regions to prevent unwanted DNA methyltransferase activity and by converting 5mC to 5hmC and further to 5 carboxylcytosine 5 caC through hydroxylase activity It was shown that genomic 5hmC level correlates to TET hydroxylase activity In addition TET1 was shown to have dual functions in transcription activation and repression by binding different target genes in ES cells TET1 is also a fusion partner of the MLL gene in acute myeloid leukemia and is considered an oncoprotein TET2 is found to be frequently muta
12. h 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 The DS solution will turn blue in the presence of sufficient hydroxymethylated DNA Add 50 ul of SS to each well to stop enzyme reaction when the color in the positive control wells turns medium blue The color 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 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 TET Activity Calculation Calculate the average duplicate readings for sample wells and blank wells Calculate TET activity or inhibition using the following formulas For simple calculation Sample OD Blank OD TET Activity OD min ng X 1000 Protein Amount ug x min
13. m 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 room 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 O Adjustable pipette or multiple channel pipette O Multiple channel pipette reservoirs O Aerosol resistant pipette tips 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 3086 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 oO o o O Oo o GENERAL PRODUCT INFORMATION Parafilm M or aluminum foil Quality Control Each lot of Epigenase 5mC Hydroxylase TET 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 u
14. nit 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 coat 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 Thus only use the User Guide that was supplied with the kit when using that kit Usage Limitation The Epigenase 5mC Hydroxylase TET Activity Inhibition Assay Kit Colorimetric is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The Epigenase 5mC Hydroxylase TET Activity Inhibition Assay Kit Colorimetric and methods of use contain proprietary technologies by 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 modification in phenotype and gene expression has been recognized widely 5 hydroxym
15. ows the specific activity of TET hydroxylases to be quantified e Strip microplate format makes the assay flexible manual or high throughput analysis 96 assays PRINCIPLE amp PROCEDURE In this assay a methylated substrate is stably coated onto microplate wells Active TETs bind to the substrate and convert methylated substrate to hydroxymethylated products The TET converted hydroxymethylated products can be recognized with a specific antibody The ratio or amount of hydroxymethylated products which is 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 TET enzyme is in turn 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 3086 0D450 nm OD450 nm 0 r r r r 1 0 r r r r 1 0 100 200 300 400 500 0 0 4 0 8 12 16 2 TET1 ng TET assay standard ng Fig 1 Demonstration of high sensitivity and specificity of Fig 2 Illustrated standard curve the TET1 activity inhibition assay achieved by using generated with the TET assay recombinant TET1 with the Epigenase 5mC Hydroxylase standard TET Activity Inhibition Assay Kit Colorimetric
16. te at 37 C for 90 min 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 Final TAB to each blank well Standard Wells Add 50 ul of Final TAB to each standard well Sample Wells Without Inhibitor Add 46 to 49 ul of Final TAB and 1 to 4 ul of nuclear extracts or purified TET enzyme to each sample well without inhibitor Total volume should be 50 ul per well Sample Wells With Inhibitor Add 41 to 44 ul of Final TAB 1 to 4 ul of nuclear extracts or purified TET 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 60 90 min Note 1 The incubation time may depend on intrinsic TET activity However in general 60 min incubation is suitable for active purifie
17. ted in leukemia and considered to act as tumor suppressor TET3 has been demonstrated to play a unique role for DNA methylation reprogramming processes in the mammalian zygote Thus activating tumor suppressor TET enzymes such as TET2 or inhibiting oncoprotein TET enzymes such as TET1 would be important in benefiting cancer diagnositcs and developing new target based cancer therapeutics However there are few methods available for detecting TET hydroxylase activity inhibition using both nuclear extracts and purified enzymes To address this issue Epigentek developed and offers the Epigenase 5mC Hydroxylase TET Activity Inhibition Assay Kit Colorimetric The kit has the following advantages and features e Colorimetric assay with easy to follow steps for convenience and speed The entire procedure can be finished within 5 hours e Directly measures TET hydroxylase activity via a straightforward detection of TET converted hydroxymethylated products e Innovative kit composition enables background signals to be extremely low and allows the assay to be simple accurate reliable and consistent e Both cell tissue extracts and purified TET proteins can be used which allows detection of inhibitory effects of TET hydroxylase inhibitor in vivo and in vitro e Novel assay principle allows high sensitivity to be achieved The activity can be detected from as low as 20 ng of purified TET1 hydroxylase e Hydroxymethylated standard is included which all
18. tely 110 Bi County 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 3086 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 3086 48 Cat P 3086 96 Upon Receipt WB 10X Wash Buffer 14 ml 28 ml 47 TAB TET Assay Buffer 3 ml 6 ml RT TS 10 X TET Substrate 10 ul 20 ul 20 C BS Binding Solution 5 ml 10 ml RT HC5 TET Assay Standard 20 10 ul 20 ul 20 C ug ml HC6 Capture Antibody 1000 4 ul 8 ul 4 C ug ml HC7 Detection Antibody 400 8 ul 16 ul 20 C ug ml HC8 Enhancer Solution 8 ul 16 ul 20 C DS Developer Solution 5ml 10 ml 4 C SS Stop Solution 3 ml 6 ml RT Co factor 1 25 ul 50 ul 4 C Co factor 2 25 ul 50 ul 4 C Co factor 3 25 ul 50 ul 47 8 Well Assay Strips With Frame 6 12 4 C 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 HC5 HC7 and HC8 at 20 C away from light 2 Store WB HC6 DS Co factor 1 Co factor 2 Co factor 3 and 8 Well Assay Strips at 4 C away fro
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