Epigenase™ Universal SIRT Activity/Inhibition Assay Kit
Contents
1. Epigenase Universal SIRT Activity Inhibition Assay Kit Colormetric Base Catalog P 4036 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The Epigenase Universal SIRT Activity Inhibition Assay Kit Colorimetric is suitable for measuring activity inhibition of total SIRT enzyme using nuclear extracts or purified SIRT isoforms SIRTs1 7 from a broad range of species such as mammals plants fungi and bacteria in a variety of forms including but not limited to cultured cells fresh and frozen tissues For SIRT1 SIRT6 and SIRT7 nuclear extracts are required and can be prepared by using your own successful method For your convenience and the best results Epigentek offers a nuclear extraction kit Cat OP 0002 optimized for use with this kit Nuclear extracts can be used immediately or stored at 80 C for future use For SIRT2 SIRT4 and SIRT5 cytoplasmic extracts are required For SIRT3 mitocondria fractions should be used Purified enzymes can be active SIRTs from recombinant proteins or isolated from cell tissues Starting Materials Input materials can be cell extracts or purified SIRT enzymes The amount of nuclear extracts for each assay can be between 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 Internal Control A SIRT assay standard deacetylated histones is provided in this kit for the quan2. 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 OD450 nm 1 65 1 44 1 2 4 14 0 8 0 6 0 4 5 0 2 4 0 T T T T 1 0 20 40 60 80 100 SIRT1 ng Demonstration of high sensitivity of a SIRT activity assa achieved by using recombinant SIRT1 with Epigenase Universal SIRT Activity Inhibition Assay Kit Colorimetric 1 8 1 6 1 4 1 2 1 0 8 OD450 nm 0 6 0 4 0 2 0 H_ HA H_ 1 0 1 2 3 4 5 SIRT assay standard ng Illustrated standard curve generated with SIRT assay standard Page 5 Printed 2014 10 13 P 4036 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 between 0 5 ug to 20 ug 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 catalatic activity of the enzymes Cell Extraction You can use your method of choice for preparing nuclear extracts cytoplasmic or mitochondria fractions Epigentek offers a nuclear extraction kit Cat OP 0002 optimized for use with this kit for SIRT1 SIRT6 and SIRT7 assay Cell Extract or Purified SIRT Enzyme Storage Cell extract o
3. 120 min 0 75 0 15 SIRT activity X 1000 1 OD min mg 5 xX 120 110 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 8 Printed 2014 10 13 P 4036 For accurate or specific activity calculation 1 Generate a standard curve and plot OD value versus amount of HO3 at each concentration point Determine the slope as OD ng you can use Microsoft Excel statistical functions for slope calculation then calculate the amount of SIRT converted deacetylated product using the following formulas Sample OD NNC OD Deacetylated product ng Slope For inhibition calculation Inhibitor Sample OD NNC OD Inhibition 1 X 100 No Inhibitor Sample OD NNC OD SUGGESTED WORKING BUFFER AND SOLUTION SETUP Table 1 Amount of required buffers and solutions for defined assay wells Reagents 1 well 1 strip 2 strips 6 strips 12 strips 8 wells 16 wells 48 wells 96 wells Diluted WB 2 5 ml 20 ml 40 ml 120 ml 240 ml HO1 50 ul 400 ul 800 ul 2400 ul 4800 ul HO2 1 ul 8 ul 16 pl 50 ul 120 ul HO3 N A N A 1 ul optional j2ul 2 ul Diluted HO4 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted HO5 50 ul 400 ul 800 ul 2400 ul 4800 ul NAD 2 ul 16
4. 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 10 13 P 4036 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 extracted or purified Protein sample is not properly Ensure your protocol is suitable for SIRT 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 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 SIRT 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 Insufficient washing of the wells Ensure the wells are washed according to the
5. ul 4 2 0 ul 3 0 ul 2 0 ng ul 5 4 0 ul 0 0 ul 5 0 ng ul 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 10 13 Epigentek Group Inc All rights reserved Products are for research use only P 4036 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 It is advised to run replicate samples include blank and positive control to ensure that the signal generated is validated 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 Blank Wells Add 48 ul of HO1 and 1 ul of HO2 and 1 ul of NAD Total volume should be 50 ul well No NAD Control NNC Wells Add 44 to 47 ul of HO1 and 1 ul of HO2 1 ul of TSA and 1 to 4 ul of cell extracts or 1 to 4 ul of purified SIRT enzyme Total volume should be 50 ul well Standard Wells Add 49 ul of HO1 and 1 ul of Diluted HO3 to each standard well with a minimum of five wells each at different concentrations between 0 2 to 5 ng ul based on the dilution chart in Step 1d see Table 2 under the Suggested Working Buffer amp Solution Setup section as an example Sa
6. ul 32 ul 96 ul 192 ul Developer 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml Solution Stop 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml Solution SUGGESTED STRIP WELL SETUP Well Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A Blank Blank Sample Sample Sample Sample B NNC NNC Sample Sample Sample Sample c HO30 2ng HO30 2ng Sample Sample Sample Sample D HO30 5ng HO30 5ng Sample Sample Sample Sample E HO31 0ng HO31 0ng Sample Sample Sample Sample F HO32 0ng HO32 0ng Sample Sample Sample Sample 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 9 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 2019 1019 P 4036 Table 2 The suggested strip wall plate G HO3 5 ng HO3 5 ng Sample Sample Sample Sample setup for SIRT activity assay ina H Sample Sample Sample Sample Sample Sample 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 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
7. 47 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 receipt 1 Store HO2 HO3 HO5 NAD TSA and NAM at 20 C away from light 2 Store WB HO4 DS and 8 Well Assay Strips at 4 C away from light 3 Store remaining components HO1 SS 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 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 and 2 check if a blue color present in DS Developer Solution which would indicate contamination of the solution and should not be used To avoid contamination transfer the amount of DS required into a secondary container tube or vial before adding DS into the assay wells MATERIALS REQUIRED BUT NOT SUPPLIED 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 Adjustable pipette or multiple channel pipette Oo Multiple channel pipette reservoirs O Aerosol resis
8. 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 is 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 stored 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 HO3 SIRT 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 HO5 is too long The incubation time at Step 3d should not exceed 2 hours 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718
9. are often contained in enzyme solutions or tested compound solvents and 4 Less accuracte than a direct measurement of SIRT converted deacetylated products We have now added the Epigenase Universal SIRT Activity Inhibition Assay Kit Colorimetric to address this issue This kit has the following advantages e Strip microplate format makes the assay flexible and quick manual or high throughput analysis can be completed within 3 5 hours e Unique kit composition enables background signals to be very low which allows the assay to be accurate sensitive reliable and consistent e Innovative colorimetric assay measures SIRT activity inhibition by directly detecting SIRT converted deacetylated products rather than trypsin based peptide cleavage thus eliminating assay interference caused by DMSO and thiol containing chemicals trypsin and cellular lysyl endipeptidases e Both cell tissue extracts and purified SIRT enzymes can be used which allows for the detection of inhibitory effects of SIRT 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 1 ng of purified SIRT enzyme which is about 10 fold higher than that obtained by trypsin based peptide cleavage assays e A deacetylated histone standard is included which allows for the specific activity of SIRTs to be quantified e Nicotinamide a SIRT inhibitor as the positive inhibition control and trichos
10. e 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 three times with 150 ul of the Diluted WB 1X Wash Buffer each time 3 Antibody Binding and Signal Enhancing a Add 50 ul of the Diluted HO4 to each well then cover with Parafilm M or aluminum foil and incubate at room temperature for 60 min b Remove the Diluted HO4 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 10 13 Epigentek Group Inc All rights reserved Products are for research use only P 4036 c Wash each well three times with 150 ul of the Diluted WB each time d Add 50 ul of the Diluted HOS5 to each well then cover with Parafilm M or aluminum foil and incubate at room temperature for 30 min e Remove the Diluted HO5 solution from each well f Wash each well four times with 150 ul of the Diluted WB each time Note Ensure any residual wash buffer in the wells is removed as much as possible at each wash step The wash can be done by simply pipetting the washing 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 change i
11. 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 EpiQuik Nuclear Extraction Kit SIRT Activity Inhibition Assay P 4034 Epigenase HDAC Activity Inhibition Assay Kit Colorimetric P 4035 Epigenase HDAC Activity Inhibition Assay Kit Fluorometric P 4037 Epigenase Universal SIRT Activity Inhibition Assay 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 10 13 P 4036
12. hydrolyzeacetyl lysine residues sirtuins catalyze a reaction that couples lysine deacetylation to NAD hydrolysis yielding O acetyl ADP ribose and nicotinamide Sirtuins have been implicated in influencing aging and regulating transcription apoptosis and stress resistance as well as energy efficiency and alertness during low calorie situations SIRTs are also involved in the development of human diseases including cancer diabetes and various neurological diseases For example in prostate cancer SIRT1 was found to be overexpressed It was also observed that SIRTs protect neurons in Alzheimer s disease Detection of inhibition or activation of SIRTs would be important in elucidating mechanisms of epigenetic regulation of gene activation and silencing and benefiting diagnostics and therapeutics of cancer or neurological diseases There are only a couple of methods used for detecting SIRT activity inhibition These methods are based on the measurement of the deacetylated histone cleavage by lysyl endopeptidase or trypsin and have significant weaknesses 1 nuclear extracts from cell tissues cannot be used because of interfering by lysyl endipeptidases from extracts 2 Trypsin sensitive SIRT inhibitiors or activators are not suitable for testing with these methods as trypsin digestion can lead to false positives when trypsin inhibitors or activators present in the compound library 3 High interference by DMSO and thiol containing chemicals which
13. mple Wells Without Inhibitor Add 42 to 45 ul of HO1 1 ul of HO2 1 ul of TSA 1 ul of NAD and 1 to 4 ul of nuclear extracts or 1 to 4 ul of purified SIRT enzyme Total volume should be 50 ul well Sample Wells With Inhibitor Add 36 to 39 ul of HO1 1 ul of HO2 1 ul of TSA 1 ul of NAD and 1 to 4 ul of nuclear extracts or 1 to 4 ul of purified HDAC enzyme and 5 ul of inhibitor solution Total volume should be 50 ul well Note 1 Follow the suggested well setup diagrams under Suggested Working Buffer amp Solution Setup section 2 It is recommended to use 2 ug to 10 ug of cell extract per well or 10 ng to 200 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 HO1 at a 1 10 ratio e g add 0 5 ul of inhibitor to 4 5 ul of HO1 so that the original solvent of the inhibitor can be reduced to 1 of the reaction solution or less The SIRT inhibitor nicotinamide NAM included in the kit can be used as a control inhibitor Tightly cover strip plate with Adhesive Covering Film to avoid evaporation and incubate at 37 C for 60 90 min Note 1 The incubation time may depend on intrinsic SIRT activity However in general 60 min incubation is suitable for active purified SIRT enzyme and 90 min incubation is required for nuclear extract 2 The Adhesiv
14. n the sample wells and control wells The DS solution will turn blue in the presence of sufficient deacetylated products b Add 100 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 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 strip well microplate frame does not fit in the microplate reader transfer the solution to a standard 96 well microplate 5 SIRT Activity Calculation a Calculate average duplicate readings for sample wells and blank wells b Calculate SIRT activity or inhibition using the following formulas For simple calculation Sample OD NNC OD SIRT Activity OD nin mg 5 _ x 1000 Protein Amount ug x min Protein amount added into the reaction at step 2e incubation time minutes at step 2g Example calculation Average OD450 of sample is 0 75 Average OD450 of NNC is 0 15 Protein amount is 5 pg Incubation time is 2 hours
15. pplication Intellectual Property The Epigenase Universal SIRT Activity Inhibition Assay Kit Colorimetric and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW Acetylation of the epsilon amino group of specific lysine residues contained in core histones is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes The acetylation of histone by histone acetyltransferases HAT seems to be of particular significance as it is associated with active regions of the genome In contrast histone deacetylation by histone deacetylase HDAC leads to transcription repression So far at least 4 classes of HDACs have been identified Class HDACs include 1 2 3 and 8 Class II HDACs are comprised of 4 5 6 7 9 and 10 Class Ill enzymes known as the sirtuins require NAD cofactors and include SIRTs 1 7 Class IV enzymes which contains only HDAC11 has features of both Class and Il 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 10 13 P 4036 CoA HATs CoASH ee acetyl lysine X Histone X lysine X Histone CHCOOH SIRT H2O NAD Fig 1 Histone acetylation and deacetylation catalyzed by HATs and HDACs Unlike other known protein deacetylases which simply
16. r purified SIRT enzyme should be stored in aliquot at 80 C until use 1 Working Buffer and Solution Preparation Prepare 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 Prepare Diluted HO4 Capture Antibody Solution Dilute HO4 Capture Antibody with Diluted WB 1X Wash Buffer at a ratio of 1 1000 add 1 ul of HO4 to 1000 ul of Diluted WB 1X Wash Buffer 50 ul of Diluted HO4 will be required for each assay well Prepare Diluted HO5 Detection Antibody Solution Dilute HO5 Detection Antibody with Diluted WB 1X Wash Buffer at a ratio of 1 2000 add 1 ul of HO5 Detection Antibody to 2000 ul of Diluted WB 1X Wash Buffer 50 ul of Diluted HO5 will be required for each assay well Prepare Diluted HO3 Standard Solution Suggested Standard Curve Preparation First dilute HO3 Assay Standard with HO1 Assay Buffer to 5 ng ul by adding 1 ul of HOS to 9 ul of HO1 Then further prepare five concentrations by combining the 5 ng ul diluted HO3 with HO1 into final concentrations of 0 2 0 5 1 2 and 5 ng ul according to the following dilution chart Resulting HO3 Tube HO3 5ng pl HOt Concentration 1 1 0 ul 24 0 ul 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
17. tant pipette tips O Microplate reader capable of reading absorbance at 450 nm Page 2 Printed 2014 10 13 P 4036 1 5 ml microcentrifuge tubes Incubator for 37 C incubation Plate seal Distilled water Nuclear extract or purified enzymes Oo Oo Oo Oo Oo Oo GENERAL PRODUCT INFORMATION Parafilm M or aluminum foil Quality Control Each lot of the Epigenase Universal SIRT 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 Universal SIRT Activity Inhibition Assay Kit Colorimetric is for research use only and is not intended for diagnostic or therapeutic a
18. tatin A TSA an inhibitor of HDACI II used to block HDAC activity are both included 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 10 13 Epigentek Group Inc All rights reserved Products are for research use only P 4036 PRINCIPLE amp PROCEDURE The Epigenase Universal SIRT Activity Inhibition Assay Kit Colorimetric contains all reagents necessary for the measurement of SIRT activity inhibition In this assay an acetylated histone SIRT substrate is stably coated onto the microplate wells Active SIRTs bind to the substrate and removes acetyl groups from the substrate The SIRT deacetylated products can be recognized with a specific antibody The ratio or amount of deacetylated products which is proportional to the enzyme activity can then be colorimetrically measured by reading the absorbance in a microplate spectrophotometer at 450 nm The activity of the SIRT enzyme is proportional to the OD intensity measured Start with nuclear extract or purified SIRT enzyme Incubate with substrate and assay buffer for 90 minutes l Wash wells then add capture antibody Wash wells then add detection antibody Add color developing solution for color develop ment then measure absorbance Schematic procedure of Epigenase Universal SIRT Activity Inhibition Assay Kit Colorimetric
19. tification of SIRT enzyme activity Because SIRT 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 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 10 13 Epigentek Group Inc All rights reserved Products are for research use only P 4036 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 4036 48 Cat P 4036 96 Upon Receipt WB 10X Wash Buffer 14 ml 28 ml 47 HO1 SIRT Assay Buffer 4ml 8 ml RT HO2 SIRT Substrate 50 X 60 ul 120 ul 20 C HO3 SIRT Assay Standard 50 ug ml 10 ul 20 ul 20 C HO4 Capture Antibody 1000 X 5 ul 10 pl 4 HO5 Detection Antibody 2000 X 6 ul 12 ul 20 C NAD SIRT Co factor 50 X 50 ul 100 ul 20 C TSA HDAC inhibitor 50 uM 50 ul 100 u 20 C NAM SIRT Inhibitor 50 mM 40 ul 80 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
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