EpiQuik™ HDAC2 Assay Kit
Contents
1. EpiQuik HDAC2 Assay Kit Colorimetric Base Catalog P 4006 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik HDAC2 Assay Kit is very suitable for measuring HDAC2 levels from various fresh tissues and cultured mammalian cells 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 03 Epigentek Group Inc All rights reserved Products are for research use only P 4006 KIT CONTENTS Contents A8 assays 96 assays P 4006 48 P 4006 96 HB1 10X Wash Buffer 11 ml 22 ml HB2 HDAC Assay Buffer l ml 2 mi HB3 Blocking Buffer 10 ml 20 ml HB4 Capture Antibody 200 ug ml 13 ul 26 ul HB5 Detection Antibody 200 ug ml 10 ul 20 ul HB6 Developing Solution 6 ml 12 ml HB7 Stop Solution 3 ml 6 ml HDAC2 Control 100 ng ul 16 ul 32 ul 8 Well Assay Strip with Frame 6 12 User Guide 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 the first part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt 1 Store HB5 and HDAC2 Control at 20 C 2 Store HB1 HB3 HB4 HB6 and 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 p2. can be quantitied through an HRP conjugated secondary antibody color development system and is proportional to the intensity of the color development 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 10 03 Epigentek Group Inc All rights reserved Products are for research use only P 4006 ij Nuclear extract Incubate with substrate and assay buffer for 60 min Add capture antibody Add detection antibody Add developing solution for developing color and measure absorbance AAFAA Schematic Procedure for Using the EpiQuik HDAC2 Assay Kit PROTOCOL 1 Prepare nuclear extracts by using your own successful method For your convenience and the best results Epigentek offers a nuclear extraction kit Cat No OP 0002 1 optimized for use in the EpiQuik series Nuclear extracts can be used immediately or stored at 80 C for future use 2 Determine the number of strip wells required the strip wells can be broken off Leave these strip wells in the plate frame remaining unused strips can be placed back in the bag Seal the bag tightly and store at 4 C Dilute HB1 with distilled water pH 7 2 to 7 5 ata 1 10 ratio ex 1 ml of HB1 9 ml of distilled water in order to create 1X HB 3 Adjust protein concentration to 0 4 1 ug ul with HB2 and add 10 ul 4 10 ug of the protein solution into the central
3. HB6 to each well and incubate at room temperature for 2 10 minutes away from light Monitor the color development in the sample and standard wells until it starts turning medium blue Add 50 ul of HB7 to each well and read absorbance on microplate reader at 450 nm Calculate HDAC2 level HDAC2 level OD ml sample OD blank OD x sample dilution For an accurate calculation plot OD value versus amount of HDAC2 control and determine the slope as delta OD ng Calculate the amount of HDAC2 using the following formula 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 OD sample blank Amount ng mg protein _ x 1000 Slope X Protein amount ug Nuclear extract added into sample wells at Step 3 TROUBLESHOOTING No Signal for Both the Positive Control and the Samples Reagents are added incorrectly Check if reagents are added in the proper order and if any steps in the procedure may have been omitted by mistake The well is not completely dried Ensure the well is incubated without humidity and dried before adding HB3 Blocking Buffer Page 5 Printed 2014 10 03 P 4006 The well is incorrectly washed before protein coating Incubation time and temperature are incorrect Ensure the well is not w
4. area of each well Spread the solution out over the bottom of the strip wells by pipetting the solution up and down several times Incubate the strip wells at 37 C without humidity for 90 minutes to evaporate the solution and completely dry the wells For the blank add 5 ul of HB2 to the wells For the positive control dilute HDAC2 Control to 1 20 ng ul with HB2 and add 10 ul 10 200 ng of the diluted HDAC2 Control solution to the wells 4 Add 150 ul of HB3 to the dried wells and incubate at 37 C for 30 45 minutes 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 03 Epigentek Group Inc All rights reserved Products are for research use only P 4006 11 12 Aspirate and wash each well three times with 150 ul of 1X HB1 each time Dilute HB4 at a 1 200 ratio to 1 ug ml with 1X HB1 Add 50 ul of diluted HB4 to each well Incubate the samples at room temperature for 60 minutes on a orbital shaker 50 100 rpm Aspirate and wash each well four times with 150 ul of 1X HB1 each time Dilute HB5 at a 1 1000 ratio to 0 2 ug ml with 1X HB1 Add 50 ul of diluted HB5 to each strip well and incubate at room temperature for 30 minutes Aspirate and wash each well four times with 150 ul of 1X HB1 each time In the last wash allow 1X HB1 to sit in the wells for 3 minutes before finally aspirating Add 100 ul of
5. ashed before adding the positive control or protein extracts Ensure the incubation time and temperature described in the protocol are followed correctly No Signal or Very Weak Signal For Only the Positive Control The HDAC2 control protein is insufficiently added to the well The positive control is degraded due to incorrect storage No Signal for Only the Sample The protein amount is added into the well insufficiently Nuclear extracts are incorrectly stored High Background Present for the Blank The well is not washed enough Contaminated by the positive control Overdevelopment RELATED PRODUCTS Ensure sufficient amount of control protein is added Follow the guidance in the protocol for storage of the positive control Ensure the extract contains a sufficient amount of protein Ensure the nuclear extracts are stored at 80 C Check if wash at each step is performed according to the protocol Ensure the well is not contaminated from add ing the control protein or from using control protein contaminated tips Decrease development time in step 10 P 4002 EpiQuik HDAC Activity Inhibition Assay Kit Colorimetric P 4005 EpiQuik HDAC1 Assay Kit P 4007 EpiQuik HDAC8 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 res
6. earch use only Page 6 Printed 2014 10 03 P 4006
7. is cell cycle arrest and differentiation in cancer cells HDAC inhibitors are currently being developed as potential anticancer agents Three distinct families of HDACs have been described comprising a group of at least 20 proteins in humans HDAC2 is a class histone deacetylase containing 488 amino acid residues HDAC2 has been shown to interact directly with transcription factors and has been shown to deacetylate histone proteins H3 and H4 The major assay for measuring the expression or amount of HDAC2 protein currently is Western blot This method requires electrophoresis and transfer process which makes the assay inconvenient time consuming and has low throughput The EpiQuik HDAC2 Assay Kit addresses these problems by using a unique procedure to measure the amount of HDAC2 The kit has the following features e The fastest procedure which can be finished within 3 hours e Innovative colorimetric assay to semi quantitatively measure HDAC2 amount without the need for electrophoresis e Strip microplate format makes the assay flexible manual or high throughput analysis e Simple reliable and consistent assay conditions PRINCIPLE amp PROCEDURE The EpiQuik HDAC2 Assay Kit is designed for measuring total HDAC2 amount from tissues or cells In an assay with this kit the nuclear proteins containing HDAC2 are stably coated on the strip wells The HDAC2 is recognized with a high affinity specific antibody The amount of HDAC2
8. roperly Note Check if wash buffer HB1 contains salt precipitates before using If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED O Orbital shaker O Pipettes and pipette tips O Microplate reader O 1 5 ml microcentrifuge tubes GENERAL PRODUCT INFORMATION Quality Control Epigentek guarantees the performance of all products in the manner described in our product instructions Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 2 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 10 03 Epigentek Group Inc All rights reserved Products are for research use only P 4006 Usage Limitation The EpiQuik HDAC2 Assay Kits are for research use only and are not intended for diagnostic or therapeutic application Intellectual Property EpiQuik is a trademark of Epigentek Group Inc A BRIEF OVERVIEW Histone deacetylases HDACs play a critical role in transcriptional repression of gene expression in eukaryotic cells through 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 apoptos
Download Pdf Manuals
Related Search