EpiQuik™ HDAC3 Assay Kit
Contents
1. No signal or weak signal only in sample wells Protein sample is not properly extracted or purified Ensure your protocol is suitable for histone protein extraction For the best results it is advised to use Epigentek s Nuclear extraction Kit Cat No OP 0002 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 10 31 P 4040 Sample amount added into the wells is insufficient Ensure a sufficient amount of 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 months for nuclear extracts Little or no HDAC3 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 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 stop2. EpiQuik HDAC3 Assay Kit Colorimetric Base Catalog P 4040 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik HDAC3 Assay Kit is designed for measuring HDAC3 protein amounts quantitatively from fresh tissue and cultured cells of human and mouse 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 31 Epigentek Group Inc All rights reserved Products are for research use only P 4040 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 4040 48 Cat P 4040 96 Upon Receipt WB 10X Wash Buffer 12 ml 25 ml 47 AB Assay Buffer 5 ml 10 ml 47 BB Blocking Buffer 10 ml 20 ml 47 CA Capture Antibody 1000 ug ml 5 ul 8 ul 4 DA Detection Antibody 200 ug ml 6 ul 10 pl 20 C ES Enhancer solution 6 ul 10 ul 20 C DS Developing Solution 6 ml 12 ml 47 SS Stop Solution 6 ml 11 ml RT HDAC3 control 100 pg ml 8 ul 16 ul 20 C 8 Well Assay Strips With Frame 6 12 47 User Guide 1 1 RT For maximum recovery of the products centrifuge the original vial after thawing prior to opening the cap SHIPPING amp STORAGE The kit is shipped in two parts one part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt 1 Store DA ES and HDAC3 Control at 20 C away from light 2 Store
3. following chart Resulting ee HDAC3 Concentration 1 0 5 ul 9 5 ul 1 ng ul 2 0 5pl 4 5 ul 2 ng ul 3 1 0 pl 3 0 ul 5 ng ul 4 2 0ul 2 0 ul 10 ng ul 5 4 0 ul 0 0 pI 20 ng ul Note Keep each of the diluted solutions except 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 The lower concentration point ex 0 5 ng ul can be also added if needed 2 HDAC3 Binding a Predetermine the number of strip wells required for your experiment It is advised to run replicate samples include blank and positive controls 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 b Blank Wells Add 100 ul of AB to each blank well c Standard Wells Add 99 ul of AB and 1 ul of Diluted HDAC3 control to each standard well with a minimum of five wells each at a different concentration between 1 and 20 ng ul based on the dilution chart in Step 1e see Table 2 in the Suggested Strip Well Setup section as an example d Sample Wells Add 94 to 98 ul of AB and 2 to 6 ul of your nuclear extracts to each sample well Total volume should be 100 ul per well Note 1 Follow the diagram in the Suggested Strip Well Setup section 2 It is recommended to use 2 ug to 4 ug of nuclear extrac
4. 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 Histone Deacetylase Activity Inhibition Assay Epigenase HDAC Activity Inhibition Direct Assay Kit Epigenase HDAC Activity Inhibition Direct Assay Kit Fluorometric Epigenase Universal SIRTActivity Inhibition Assay Kit Epigenase Universal SIRT Activity Inhibition Assay Kit Fluorometric P 4034 P 4035 P 4036 P 4037 P 4005 P 4006 P 4042 P 4044 P 4046 P 4048 P 4007 P 4050 P 4052 P 4054 EpiQuik HDAC1 Assay Kit EpiQuik HDAC2 Assay Kit EpiQuik HDAC4 Assay Kit EpiQuik HDAC5 Assay Kit EpiQuik HDAC6 Assay Kit EpiQuik HDAC7 Assay Kit EpiQuik HDAC8 Assay Kit EpiQuik HDACS9 Assay Kit EpiQuik HDAC10 Assay Kit EpiQuik HDAC11 Assay 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 Products are for research use only Page 10 Printed 2014 10 31 P 4040
5. WB AB BB CA DS and the 8 Well Assay Strips at 4 C away from light 3 Store all other components at room temperature The kit is stable for up to 6 months from the shipment date when stored properly Note 1 Check if wash buffer WB contains salt precipitates before using If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved 2 check if a blue color is present in DS Developing 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 oO 0 OF O 0 O 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 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 10 31 P 4040 O Distilled water O Nuclear extracts or purified HDAC3 enzyme O Parafilm M or aluminum foil GENERAL PRODUCT INFORMATION Quality Control Each lot of the EpiQuik HDAC3 Assay Kit is tested against predetermined specification
6. ated Control Sample OD Blank OD 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 7 Printed 2014 10 31 P 4040 Example calculation Average OD450 of treated sample is 0 5 Average OD450 of untreated control is 0 9 Average OD450 of blank is 0 1 0 5 0 1 HDAC3 change x 100 50 For Detailed Quantification 0 9 0 1 1 Generate a standard curve and plot OD value versus amount of HDAC3 control standard 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 HDAC3 using the following formulas i Sample OD Blank OD HDAC3 ng mg protein __ x 1000 Slope x Protein Amount ug Nuclear extract added into sample wells at Step 2d SUGGESTED BUFFER AND SOLUTION SETUP Table 1 Approximate amount of required buffers and solutions for defined assay wells based on the protocol 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 AB 100 ul 800 ul 1600 ul 4900 ul 9600 ul BB 0 15 ml 1 2 ml 2 5 ml 7 5 ml 14 5 ml HDAC3 control N A N A 2 uL optional 4 ul 4 ul Dil
7. djust 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 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 50 ul of Diluted CA will be required for each 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 50 ul of Diluted DA will be required for each assay well 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 5 Tel 1 877 Epigentek Group Inc All rights reserved Products are for research use only P 4040 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 10 31 j bb co sa g see A d Prepare Diluted ES Enhancer Solution Dilute ES Enhancer Solution with Diluted WB at a ratio of 1 5000 i e add 1 ul of ES to 5000 ul of Diluted WB About 50 ul of this Diluted ES will be required for each assay well e Prepare Diluted HDAC3 Control Standard Suggested Standard Curve Preparation First dilute HDAC3 control with AB to a concentration of 20 ng ul by adding 2 ul of HDAC3 control to 8 ul of AB Then further prepare concentration points of 1 2 5 10 and 20 ng ul according to the
8. gentek Group Inc All rights reserved Products are for research use only P 4040 e Very fast procedure which can be finished within 4 hours e Innovative colorimetric assay to quantitatively measure HDAC3 protein amount without the need for electrophoresis e High sensitivity and specificity HDAC3 specific detection with a detection limit as low as 0 2 ng of HDAC3 protein e Strip microplate format makes the assay flexible manual or high throughput analysis e HDAC3 control is included which allows for the HDAC3 protein amount of the sample to be properly quantified e Simple reliable and consistent assay conditions PRINCIPLE amp PROCEDURE The EpiQuik HDAC3 Assay Kit Colorimetric is designed for measuring total HDAC3 protein amount from tissues or cells In an assay with this kit the unique HDAC affinity substrate is stably coated on the strip well The sample is added into the well and HDAC3 proteins contained in the sample bind to the substrate The bound HDAC3 can be recognized with an HDAC3 specific antibody and colorimetrically quantified through an ELISA like reaction The amount of HDAC3 is proportional to the intensity of color development Start with nuclear extract or purified enzyme Incubate with substrate and assay buffer for 90 minutes Wash wells then add affinity antibody Wash wells then add detection antibody and enhancer solution lt NER X Add c
9. 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 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 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 HDAC3 control 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 DA is too long The incubation time at Step 3d should not exceed 90 min Over development of color Decrease the development time in Step 4a before adding SS Stop Solution in Step 4b
10. le arrest and differentiation in cancer cells HDAC inhibitors are currently being developed as potential anti cancer agents Three distinct families of HDACs have been described comprising a group of at least 20 proteins in humans HDACS3 is a class histone deacetylase containing 428 amino acid residues HDAC3 acts on the chromatin via the formation of large multiprotein complexes that affect gene transcription and correlate with epigenetic repression Also through interaction with numerous transcription factors HDAC3 regulates many biological processes including cell cycle progression HDACS3 is the most highly expressed class HDAC throughout the brain including the hippocampus and is a critical negative regulator of long term memory formation HDAC3 is also overexpressed in many different cancer types such as colon cancer and breast cancer Western blot is currently the most prominent assay technique for measuring the expression or amount of HDAC3 protein Yet this traditional method requires electrophoresis and transfer processes which make the assay inconvenient time consuming and low throughput The EpiQuik HDAC3 Assay Kit addresses these problems by using a unique procedure to measure the amount of HDAC3 proteins The kit has the following features 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 10 31 Epi
11. olor developing solution for color develop ment then measure absorbance Schematic procedure of the EpiQuik HDAC3 Assay Kit Colorimetric 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 31 Epigentek Group Inc All rights reserved Products are for research use only P 4040 OD450 nm 0 7 0 6 0 5 E 0 4 i S 0 3 0 2 0 1 i ft Ee T T T T T T T T T 1 o 1 5 10 20 0 2 4 6 8 10 12 14 16 18 20 Nuclear extract ug HDAC3 Control ng Nuclear extracts were prepared from HeLa cells and the Illustrated standard curve generated with HDAC3 control ODs generated from HDAC3 are measured 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 between 0 5 ug and 10 ug with an optimal range of 2 to 4 ug Nuclear Extraction You can use your method of choice for preparing nuclear extracts from the treated and untreated samples Epigentek also offers a nuclear extraction kit Cat OP 0002 optimized for use with this kit Nuclear extracts should be stored at 80 C in aliquots until use 1 Working Buffer and Solution Preparation Prepare Diluted WB 1X Wash Buffer 48 Assay Kit Add 13 ml of WB 10X Wash Buffer to 117 ml of distilled water and a
12. s 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 EpiQuik HDAC3 Assay Kit is for research use only and is not intended for diagnostic or therapeutic applications Intellectual Property The EpiQuik HDAC3 Assay Kit and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW Histone deacetylases HDACs play a critical role in transcriptional repression of gene expression in eukaryotic cells by catalyzing the hydrolytic removal of acetyl groups from histone lysine residues HDACs are tightly involved in cell cycle regulation cell proliferation and in the development of human cancer HDAC inhibition displays significant effects on apoptosis cell cyc
13. t per well e Cover strip well microplate with Parafilm M or aluminum foil to avoid evaporation and incubate at 37 C for 90 to 120 min f Remove the reaction solution from each well Add 150 ul of BB Blocking Buffer to each well then cover with Parafilm M or aluminum foil and incubate at 37 C for 30 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 Web www epigentek com Printed 2014 10 31 Epigentek Group Inc All rights reserved Products are for research use only P 4040 g 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 aluminum foil and incubate at room temperature for 60 min b Remove the Diluted CA solution from each well c Wash each well three times with 150 ul of the Diluted WB each time d Add 50 ul of the Diluted DA to each well then cover with Parafilm M or aluminum 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 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 i Wash each
14. uted CA 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted DA 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted ES 50 ul 400 ul 800 ul 2400 ul 4800 ul DS 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml ss 0 1 ml 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 HDAC3 quantification 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 Sample Sample Sample B HDAC3 1 ng HDAC3 1 ng 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 8 Printed 2014 10 31 P 4040 c HDAC3 2 ng HDAC3 2 ng Sample Sample Sample Sample D HDAC3 5 ng HDAC3 5 ng Sample Sample Sample Sample E HDAC3 10 ng HDAC3 10 ng Sample Sample Sample Sample F HDAC3 20 ng HDAC3 20 ng Sample Sample Sample Sample G Sample Sample Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample TROUBLESHOOTING Problem Possible Cause Suggestion No signal or weak signal in both the positive control and sample wells Reagents are added incorrectly Check
15. well five 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 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 in the sample wells and control wells The DS solution will turn blue in the presence of sufficient HDAC3 protein b Add 100 ul of SS 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 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 HDAC3 Calculation a Calculate the average duplicate readings for the sample wells and blank wells b Calculate HDAC3 change using the following formula Treated Tested Sample OD Blank OD o HDAC3 change X 100 Untre
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