Epigenase™ 5-mC Hydroxylase TET Activity/Inhibition
Contents
1. LSD1 Demethylase 40 Activity Inhibition Assay Kit Colorimetric a 30 5 20 5 10 0 T T T T T T 1 0 10 20 50 100 200 500 Tranylcypromine uM Demonstration of inhibitory effect of LSD1 inhibitor detected by Epigenase LSD1 Activity Inhibition Assay Ultra Kit LSD1 concentration 200 ng well PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials 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 Web www epigentek com Printed 2014 08 21 Epigentek Group Inc All rights reserved Products are for research use only P 3078 Input Amount The amount of nuclear extracts for each assay can be 0 5 ug to 20 ug with optimized range of 5 10 ug The amount of purified enzymes can be 5 ng to 500 ng depending on the purity and catalatic activity of the enzymes Nuclear Extraction You can use your method of choice for preparing nuclear extracts Epigentek also offers a nuclear extraction kit Cat No OP 0002 optimized for use with this kit Nuclear Extract or Purified LSD1 Storage Nuclear extract or purified LSD1 enzyme should be stored in aliquots at 80 C until use 1 Working Buffer and Solution Preparation a Prepare Diluted LD1 1X Wash Buffer 48 Assay Kit Add 13 ml of LD1 10X Wash Buffer to 117 ml of distilled water and adjust pH to 7 2 7 5 96 Assay Kit Add2. Epigenase LSD1 Demethylase Activity Inhibition Assay Kit Colorimetric Base Catalog P 3078 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The Epigenase LSD1 Demethylase Activity Inhibition Assay Kit Colorimetric is suitable for measuring LSD1 activity inhibition using nuclear extracts or purified enzymes from a broad range of species such as mammals plants fungi and bacteria in a variety of forms including cultured cells and fresh tissues Nuclear extracts can be prepared by using your own successful method For your convenience and the best results Epigentek offers a nuclear extraction kit Cat No OP 0002 that is optimized for use with this kit Nuclear extracts can be used immediately or stored at 80 C for future use Purified enzymes can be active LSD1 from recombinant proteins or isolated from cell tissues Input Material Input materials can be nuclear extracts or purified LSD1 enzymes The amount of nuclear extracts for each assay can be 0 5 ug to 20 yg with an optimal range of 5 10 ug The amount of purified enzymes can be 5 ng to 500 ng depending on the purity and catalytic activity of the enzymes Internal Control The LSD1 assay standard demethylated hsitone H3 K4 is provided in this kit for quantification of LSD1 enzyme activity Because LSD1 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 validate
3. 26 ml of LD1 10X Wash Buffer to 234 ml of distilled water and adjust pH to 7 2 7 5 This Diluted LD1 1X Wash Buffer can now be stored at 4 C for up to six months b Prepare Diluted LD5 Capture Antibody Solution Dilute LD5 Capture Antibody with Diluted LD1 1X Wash Buffer at a ratio of 1 1000 add 1 ul of LD5 to 1000 ul of Diluted LD1 1X Wash Buffer 50 ul of Diluted LD5 will be required for each assay well c Prepare Diluted LD6 Detection Antibody Solution Dilute LD6 Detection Antibody with Diluted LD1 1X Wash Buffer at a ratio of 1 2000 add 1 ul of LD6 Detection Antibody to 2000 ul of Diluted LD1 1X Wash Buffer 50 ul of Diluted LD6 will be required for each assay well d Prepare Diluted LD4 Standard Solution Suggested Standard Curve Preparation First dilute LD4 with LD2 to 5 ng ul by adding 1 ul of LD4 to 9 ul of LD2 Then further prepare five concentrations by combining the 5 ng l diluted LD4 with LD2 into final concentrations of 0 2 0 5 1 2 and 5 ng ul according to the following dilution chart Resulting LD4 Tube LD4 5 ng pl LD2 Concentration 1 1 0 ul 24 0 yul 0 2 ng ul 2 1 0 ul 9 0 ul 0 5 ng ul 3 1 0 ul 4 0 ul 1 0 ng ul 4 2 0 ul 3 0 ul 2 0 ng ul 5 4 0 ul 0 0 ul 5 0 ng ul Note Keep each of diluted solutions except Diluted LD1 1X Wash Buffer on ice until use Any remaining diluted solutions other than Diluted LD1 should be discarded if not used within the same day 2 Enzymatic R
4. All rights reserved Products are for research use only Printed 2014 08 21 P 3078 Sample OD Blank OD Demethylated product ng Slope Demethylated Product ng LSD1 Activity ng min mg _ X 1000 Protein Amount ug X min Incubation time minutes at Step 2f For inhibition calculation Inhibitor Sample OD Blank OD Inhibition 1 X 100 No Inhibitor Sample OD Blank OD SUGGESTED WORKING BUFFER AND SOLUTION SETUP Table 1 Approximate amount of required buffers and solutions for defined assay wells based on the protocol Reagents 1well 1 strip 2 strips 6 strips 12 strips 8 wells 16 wells 48 wells 96 wells Diluted LD1 2 5ml 20ml 40 ml 120 ml 240 ml LD2 50 ul 400 ul 800 ul 2400 ul 4800 ul LD3 1 ul 8 ul 16 ul 50 ul 120 ul LD4 NA NA 1 ul optional j2ul 2 ul Diluted LD5 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted LD6 50 ul 400 ul 800 ul 2400 ul 4800 ul Developer Solution 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml Stop Solution 0 1m 0 8 ml 1 6 ml 4 8 ml 9 6 ml SUGGESTED STRIP WELL SETUP Table 2 The suggested strip well plate setup for LSD1 activity assay 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 Strip 3 Strip 4 Strip 5 Strip 6 A Blank Blank Sample Sam
5. of multiple cellular processes The methylation of H3 K4 seems to be of particular significance as it is associated with active regions of the genome H3 K4 methylation was considered irreversible until the identification of a large number of histone demethylases indicated that demethylation events play an important role in histone modification dynamics So far at least 2 classes of H3 K4 specific histone demethylase LSD1 BHC110 KDM1 and JARIDs have been identified LSD1 can remove di and mono methylation from H3 K4 by using an amine oxidase reaction LSD1 is associated with complexes that function as both transcriptional inactivators and activators It demethylates mono di methyl H3 K4 when associated with the Co REST complex at neuronal genes or mono di methyl H3 K9 when associated with the androgen receptor H3 K4 N CHy H3 K4 N Ch H3 K4 NH R Reaction R R FAD FADH H 0 HCHO Onidative Hait Reacton Ret N R CH H0 0 Fig 1 Histone H3 K4 demethylation reaction catalyzed by LSD1 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 Web www epigentek com Printed 2014 08 21 Epigentek Group Inc All rights reserved Products are for research use only P 3078 LSD1 is found to be pivotal in development and differentiation For example this enzyme is required to induce skeletal muscle differentiation and mutation of drosophila LSD1 resul
6. 1 EpiQuik Nuclear Extraction Kit Histone Demethylase Activity Inhibition Assay EpiQuik Histone Demthylase H3 K9 Specific Activity Inhibition Fast Assay Kit Epigenase LSD1 Demethylase Activity Inhibition Assay Kit Fluorometric Epigenase JMJD2 Demethylase Activity Inhibition Assay Kit Colorimetric Epigenase JMJD2 Demethylase Activity Inhibition Assay Kit Fluorometric Epigenase JARID Demethylase Activity Inhibition Assay Kit Colorimetric Epigenase JARID Demethylase Activity Inhibition Assay Kit Fluorometric P 3077 P 3079 P 3080 P 3081 P 3082 P 3083 LSD1 and Methylated H3 K4 Antibodies A 3018 A 4031 A 4032 A 1004 LSD1 Polyclonal Antibody Histone H3K4 Monomethy Polyclonal Antibody Histone H3K4 Dimethyl Polyclonal Antibody Histone H3K4 Trimethyl Polyclonal Antibody 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 08 21 P 3078
7. 35 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 08 21 P 3078 Sample amount added into the wells is insufficient Ensure a sufficient amount of purified enzymes or nuclear extracts is used as indicated in Step 2 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 LSD1 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 nuclear extracts or purified enzymes Uneven color development Insufficient washing of the wells 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 solution or stop solution is added sequentially and is consistent with the order you added the other reagents e g from well A to well G or from well 1 to well 12 RELATED PRODUCTS Nuclear Extract Preparation OP 0002
8. NERAL PRODUCT INFORMATION Quality Control Each lot of the Epigenase LSD1 Demethylase 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 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 LSD1 Demethylase Activity Inhibition Assay Kit Colorimetric is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The Epigenase LSD1 Demethylase Activity Inhibition Assay Kit Colorimetric and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW Lysine histone methylation is one of the most robust epigenetic marks and is essential for the regulation
9. d 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 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 08 21 Epigentek Group Inc All rights reserved Products are for research use only P 3078 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 3078 48 Cat P 3078 96 Upon Receipt LD1 10X Wash Buffer 14 ml 28 ml 4T LD2 LSD1 Assay Buffer 4ml 8 ml RT LD3 LSD1 Substrate 50 pg ml 60 ul 120 ul 20 C LD4 LSD1 Assay Standard 50 ug ml 10 ul 20 ul 20 C LD5 Capture Antibody 1000 ug ml 5 ul 10 ul 4T LD6 Detection Antibody 400 pg ml 6 ul 12 ul 20 C LD7 LSD1 Inhibitor Tranylcypromine 1 mM 20 ul 40 ul 4T LD8 Developer Solution 5 ml 10 ml 4T LD9 Stop Solution 5 ml 10 ml RT 8 Well Assay Strips With Frame 6 12 4 Adhesive Covering Film 1 1 RT User Guide 1 1 RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped in two parts the first part at ambient room temperature and the second part on frozen ice packs at 4 C Upon rece
10. d at the appropriate temperature 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 in this User Guide for storage of LD4 LSD1 Assay Standard High background present in the blank wells Insufficient washing of wells Check if washing recommendations 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 Diluted LD6 is too long The incubation time at Step 3d should not exceed 2 hours Over development of color Decrease the development time in Step 4a before adding LD9 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 LSD1 protein extraction For the best results it is advised to use Epigentek 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 110 Bi County Blvd Ste 122 Farmingdale NY 117
11. e 2 ug to 10 ug of nuclear extract per well or 10 ng to 100 ng of purified enzyme per well 3 The concentration of inhibitors to be added into the sample wells can be varied e g 1 uM to 1000 uM However the final concentration of the inhibitors before adding to the wells should be prepared with LD2 at a 1 10 ratio e g add 0 5 ul of inhibitor to 4 5 ul of LD2 so that the original solvent of the inhibitor can be reduced to 1 of the reaction solution or less The LSD1 inhibitor Tranylcypromine LD7 included in the kit can be used as a control inhibitor Tightly cover strip well microplate with Adhesive Covering Film to avoid evaporation and incubate at 37 C for 60 120 min Note 1 The incubation time may depend on intrinsic LSD1 activity However in general 60 90 min incubation is suitable for active purified LSD1 enzymes and 90 120 min incubation is required for nuclear extracts 2 The Adhesive Covering Film can be cut to the required size to cover the strips based on the number of strips to be used Remove the reaction solution from each well Wash each well with 150 ul of the Diluted LD1 1X Wash Buffer each time for three times 3 Antibody Binding amp Signal Enhancing a Add 50 ul of the Diluted LD5 to each well then cover with Parafilm M or aluminium foil and incubate at room temperature for 60 min b Remove the Diluted LD5 solution from each well c Wash each well with 150 ul of the Diluted LD1 each time for three t
12. e 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 strip well microplate frame does not fit in the microplate reader transfer the solution to a standard 96 well microplate 5 LSD1 Activity Calculation a b Calculate the average duplicate readings for the sample wells and blank wells Calculate LSD1 activity or inhibition using the following formulas For simple calculation Sample OD Blank OD LSD1 Activity OD min ng _ X 1000 Protein Amount ug x min Protein amount ug added into the reaction at step 2d Incubation time minutes at step 2f Example calculation Average OD450 of sample is 0 65 Average OD450 of blank is 0 05 Protein amount is 5 pg Incubation time is 120 minutes 2 hours 0 65 0 05 LSD1 activity X 1000 1 OD min mg 5 X 120 For accurate or specific activity calculation 1 Generate a standard curve and plot OD value versus amount of LD4 at each concentration point 2 Determine the slope as OD ng you can use Microsoft Excel statistical functions for slope calculation then calculate the amount of LSD1 converted demethylated product using the following formulas 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 Epigentek Group Inc
13. eaction a Predetermine the number of strip wells required for your experiment It is advised to run replicate samples include blank and positive control to ensure that the signal generated is validated Carefully 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 Web www epigentek com Printed 2014 08 21 Epigentek Group Inc All rights reserved Products are for research use only P 3078 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 Blank Wells Add 49 ul of LD2 and 1 ul of LD3 to each blank well Standard Wells For a standard curve add 49 ul of LD2 and 1 ul of Diluted LD4 standard solution to each standard well with a minimum of five wells each at a different concentration between 0 2 to 5 ng ul based on the dilution chart in Step 1d see Table 3 as an example Sample Wells Without Inhibitor Add 45 to 48 ul of LD2 1 ul of LD3 and 1 to 4 ul of your nuclear extracts or 1 to 4 ul of your purified LSD1 enzyme to each sample well without inhibitor Total volume should be 50 ul per well Sample Well With Inhibitor Add 40 to 43 ul of LD2 1 ul of LD3 1 to 4 ul of your nuclear extracts or 1 to 4 ul of your purified LSD1 enzyme and 5 ul of inhibitor solution Total volume should be 50 ul per well Note 1 Follow the suggested well setup diagrams 2 It is recommended to us
14. imes d Add 50 ul of the Diluted LD6 to each well then cover with Parafilm M or aluminium foil and incubate at room temperature for 30 min e Remove the Diluted LD6 solution from each well f Wash each well with 150 ul of the Diluted LD1 each time for four times Note Ensure any residual wash buffer in the wells is removed as much as possible at each wash step 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 08 21 Epigentek Group Inc All rights reserved Products are for research use only P 3078 4 Signal Detection a Add 100 ul of LD8 to each well and incubate at room temperature for 1 to 10 min away from light Begin monitoring color change in the sample wells and control wells The LD8 solution will turn blue in the presence of sufficient demethylated products Add 100 ul of LD9 to each well to stop enzyme reaction when color in the positive control wells turns medium blue The color will change to yellow after adding LD9 and the absorbance should be read on a 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 plat
15. ipt 1 Store LD3 LD4 and LD6 at 20 C away from light 2 Store LD1 LD5 LD7 LD8 and 8 Well Assay Strips at 4 C away from light 3 Store remaining components LD2 LD9 and Adhesive Covering Film at room temperature away from light All components of the kit are stable for 6 months from the date of shipment when stored properly Note 1 Check if LD1 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 and 2 check if a blue color is present in LD8 Developer Solution which would indicate contamination of the solution and should not be used To avoid contamination transfer the amount of LD8 required into a secondary container tube or vial before adding LD8 into the assay wells MATERIALS REQUIRED BUT NOT SUPPLIED O Adjustable pipette or multiple channel pipette Aerosol resistant pipette tips 1 5 ml microcentrifuge tubes 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Multiple channel pipette reservoirs Microplate reader capable of reading absorbance at 450 nm 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 08 21 P 3078 Incubator for 37 C incubation Distilled water Nuclear extract or purified enzymes O OF 0 O0 Parafilm M or aluminum foil GE
16. ple Sample Sample B LD40 2ng LD40 2ng Sample Sample Sample Sample c LD40 5ng LD40 5ng Sample Sample Sample Sample D LD4 1 0ng LD41 0ng Sample Sample Sample Sample E LD4 2 0 ng LD42 0ng Sample Sample Sample Sample F LD45 0ng LD45 0ng 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 08 21 TROUBLESHOOTING Problem Possible Cause Suggestion No signal or weak signal in both the positive control 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 well is incorrectly washed before enzyme reaction Ensure the well is not washed prior to adding the positive control and sample Incubation time and temperature are incorrect Ensure the incubation time and temperature described in the protocol are followed correctly Incorrect absorbance reading Check if appropriate absorbance wavelength 450 nm is used Kit was not stored or handled properly Ensure all components of the kit were store
17. r the measurement of LSD1 activity inhibition In this assay di methylated histone H3 K4 LSD1 substrate is stably coated onto the strip wells Active LSD1 binds to the substrate and removes methyl groups from the substrate The LSD1 demethylated products can be recognized with a specific antibody The ratio or amount of demethylated products which is proportional to enzyme activity can then be colorimetrically measured by reading the absorbance in a colorimetric microplate reader at a wavelength of 450 nm The activity of LSD1 enzyme is 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 08 21 Epigentek Group Inc All rights reserved Products are for research use only P 3078 j ob co sa g see A 1 25 Start with nuclear extract or purified LSD1 enzyme Incubate with substrate and assay buffer for 90 minutes 0D450 nm 0 T T T T 1 0 50 100 150 200 250 Wash wells then add LSD1 ng capture antibody Demonstration of high sensitivity of LSD1 activity assay achieved by using recombinant LSD1 with Epigenase LSD1 Activity Inhibition Assay Ultra Kit Wash wells then add detection antibody 100 90 5 80 5 Add color developing 70 J solution for color development then S5 604 measure absorbance ke 507 Schematic procedure of Epigenase
18. ts in tissue specific defect in development through disrupting H3 K4 methylation LSD1 is also shown to participate in regulation of chromatin remodeling cell death and global DNA methylation More importantly LSD1 is found to be involved in some pathological processes such as cancer and inflammatory diseases For example expression of LSD1 is observed in cancer and LSD1 triggers MYC and E2F mediated transcription in cancer cells Detection of activity and inhibition of LSD1 would be important in elucidating mechanisms of epigenetic regulation of gene activation and silencing and benefiting cancer diagnostics and therapeutics There are only a couple of methods used for detecting LSD1 activity inhibition These methods are based on the measurement of H20 or formaldehyde release a by product of LSD1 enzymatic reaction and have significant weaknesses including 1 Large amount at yg level of substrate and enzyme are required 2 Nuclear extracts from cell tissues can not be used 3 Redox sensitive LSD1 inhibitiors are not suitable for testing with these methods 4 Highly interfered by DMSO and thiol containing chemicals which are often contained in enzyme solution or tested compound solvents and 5 Less accuracy than direct measurement of LSD1 converted demethylated product These problems were averted with the earlier EpiQuik Histone Demethylase LSD1 Activity Inhibition Assay Kit a popular assay method for LSD1 activity inhibition We ha
19. ve now added an improved version the Epigenase LSD1 Activity Inhibition Assay Kit Colorimetric This latest method retains the simplicity rapidness high throughput and non radioactivity featured in the previous version and has the following advantages e Strip well microplate format makes the assay flexible and quick manual or high throughput analysis that can be completed within 3 hours e Enhanced kit composition enables background signals to be extremely low which allows the assay to be more accurate sensitive reliable and consistent e Innovative colorimetric assay directly measures LSD1 activity by a straightforward detection of LSD1 converted demethylated product rather than by products Thus it eliminates assay interferences caused by thiol containing chemicals such as DTT GSH and 2 mercaptoethanol e Both cell tissue extracts and purified LSD1 can be used which allows for the detection of inhibitory effects of LSD1 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 5 ng of purified LSD1 enzyme which is about 20 fold higher than that obtained by H O formaldehyde release based LSD1 assays e Demethylated H3 K4 standard is included which allows the specific activity of LSD1 to be quantified PRINCIPLE amp PROCEDURE The Epigenase LSD1 Demethylase Activity Inhibition Assay Kit Colorimetric contains all reagents necessary fo
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