EpiQuik™ Global Histone H3 Phosphorylation (Ser28
Contents
1. EpiQuik Global Histone H3 Phosphorylation Ser28 Assay Kit Fluorometric Base Catalog P 7005 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Global Histone H3 Phosphorylation Ser28 Assay Kit Fluorometric is suitable for specifically measuring global histone H3 phophorylation at ser28 using a variety of mammalian cells human mouse etc including fresh and frozen tissues and cultured adherent and suspension 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 2015 04 02 Epigentek Group Inc All rights reserved Products are for research use only P 7005 KIT CONTENTS Components 48 assays 96 assays P 7005 48 P 7005 96 F1 10X wash buffer 10 ml 20 ml F2 antibody buffer 6 ml 12 ml F3 detection antibody 1 mg ml 5 pl 10 ul F4 fluoro developer 12 ul 24 ul F5 fluoro enhancer 12 ul 24 ul F6 fluoro dilution 4 ml 8 ml Standard control 100 ug ml 10 ul 20 ul 8 well sample strips with frame 4 9 8 well standard control strips 2 3 User guide For maximum recovery of the products centrifuge the original vial prior to opening the cap SHIPPING amp STORAGE Upon receipt store F3 F4 and standard control at 20 C away from light Store all other components at 4 C away from light The components of the kit are stable for up to 6 months from the date of shipm2. Mix and cover the strip wells with Parafilm M and incubate at room temperature for 1 2 hours w 4 Aspirate and wash the wells three times with 150 ul of diluted F1 each time 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 2015 04 02 Epigentek Group Inc All rights reserved Products are for research use only P 7005 are rev oo d ll i 5 Dilute F3 at 1 1000 ratio to 1 ug ml with F2 Add 50 ul of diluted F3 to each well and incubate at room temperature for 60 min on an orbital shaker 100 rpm 6 Aspirate and wash the wells six times with 150 ul of diluted F1 each time 7 Prepare fluoro development solution by adding 1 ul of F4 and 1 ul of F5 into each 400 ul of F6 Add 50 ul of fluoro development solution into the wells and incubate at room temperature for 1 5 min away from light The color in the standard wells containing the higher concentrations may turn slightly pink during this period Measure and read fluorescence on fluorescence microplate reader at 530 590 nm Note If the strip well frame does not fit the fluorescence reader transfer the solution to a standard 96 well microplate and read fluorescence at 530 590ey nm 8 Calculate histone H3 phospho ser28 RFU treated tested sample blank Phospho ser28 2 x 100 RFU untreated control sample blank For the amount quantific
3. 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 2015 04 02 Epigentek Group Inc All rights reserved Products are for research use only P 7005 Incubation time and temperature are incorrect Ensure the incubation time and temperature described in the protocol are followed correctly No Signal or Very Weak Signal for Only the Standard Control The amount of standard control is not added into standard control wells or is added insufficiently No Signal for Only the Sample The protein sample is not properly extracted The protein amount is added into well insufficiently Protein extracts are incorrectly stored High Background Present for the Blank The well is not washed sufficiently Contaminated by the standard control Overdevelopment RELATED PRODUCTS Ensure sufficient amount of control is properly added to the standard control well Ensure the procedure and reagents are correct for the nuclear protein extraction Ensure extract contains sufficient amount of protein Ensure the protein extracts are stored at 20 C or 80 C Check if wash at each step is performed according to the protocol Ensure the well is not contaminated by adding the control protein or by using control protein contaminated tips Decrease development time in Step 7 P 7001 EpiQuik In Situ Histone H3 Phosphorylati
4. ation plot RFU versus amount of standard control and determine the slope as delta RFU ng Calculate the amount of phospho ser28 using the following formula RFU sample blank Amount ng mg protein _____ x 1000 Protein ug x slope Histone extract amount added into the sample well at step 3 PLATE CONFIGURATION 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 2015 04 02 Epigentek Group Inc All rights reserved Products are for research use only P 7005 e Strip 1 3 for 96 assays or strip 1 2 for 48 assays standard wells green colored trim the standard curve can be generated with 5 8 concentration points includes blank e Example amount of standard control well Al 100 ng B1 50 ng C1 25 ng D1 12 ng E1 6 ng Fl 3 ng G1 1 5 ng H1 Ong e Strip 4 12 for 96 assays or strip 3 6 for 48 assays sample wells e Each sample or standard point can be assayed in duplicates or triplicates HISTONE EXTRACTION PROTOCOL 1 For tissues treated and untreated weigh the sample and cut the sample into small piece 1 2 mm with a scalpel or scissors Transfer tissue pieces to a Dounce homogenizer Add TEB buffer PBS containing 0 5 Triton X 100 2 mM PMSF and 0 02 NaN at 1 ml per 200 mg of tissue and disaggregate tissue pieces by 50 60 strokes Transfer homogeni
5. ent when stored properly Note Check if buffers F1 and F2 contain salt precipitates before using If so warm at room temperature or 37 C and shake the buffers until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED O Orbital shaker Pipettes and pipette tips Reagent reservoir Oo Oo O Fluorescence microplate reader O 15 ml conical tube Oo 1 5 ml microcentrifuge tubes GENERAL PRODUCT INFORMATION Usage Limitation The EpiQuik Global Histone H3 Phosphorylation Ser28 Assay Kit Fluorometric is for research use only and is not intended for diagnostic or therapeutic application Quality Control Epigentek guarantees the performance of all products in the manner described in our product instructions 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 2015 04 02 Epigentek Group Inc All rights reserved Products are for research use only P 7005 Safety Suitable lab coat disposable gloves and eye protection are required when working with the kit Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design Intellectual Property The EpiQuik Histone H3 Phosphorylation Ser28 Assay Kit and methods of use contain proprietary technologies by Epigentek EpiQuik is a trademark of Epigentek Group Inc A BRIEF OVERVIEW The phosph
6. ometric assay without the need for radioactivity electrophoresis or chromatography e Specifically captures phospho histone H3 ser28 with a detection limit as low as 0 5 ng well e Conveniently includes a control for the quantification of phosphorylated histone H3 ser28 e Strip microplate format makes the assay flexible manual or high throughput e Simple reliable and consistent assay conditions PRINCIPLE amp PROCEDURE The EpiQuik Global Histone H3 Phosphorylation Ser28 Assay Kit Fluorometric is designed for measuring global histone H3 phosphorylation at ser28 In an assay with this kit the phosphorylated histone H3 at ser28 is captured to the strip wells coated with an anti phospho histone H3 ser28 antibody The captured phospho histone H3 ser28 can then be detected with a labeled detection antibody followed by a color development reagent The ratio of phospho 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 2015 04 02 P 7005 histone H3 ser28 is proportional to the intensity of fluorescence The absolute amount of phospho histone H3 ser28 can be quantitated by comparing it to the standard control Tissue disaggregation or cell lysis z l ij Histone extracts aens Histone H3 phospho ser28 b
7. on Ser10 Assay Kit P 7002 EpiQuik Global Histone H3 Phosphorylation Ser10 Assay Kit Colorimetric P 7003 EpiQuik Global Histone H3 Phosphorylation Ser10 Assay Kit Fluorometric P 7004 EpiQuik Global Histone H3 Phosphorylation Ser28 Assay Kit Colorimetric 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 2015 04 02 P 7005
8. orylation of histone H3 at serine 28 is conserved in eukaryotes and an increase in phosphorylation has been shown to correlate with gene activation and cell growth In vitro studies have shown that phosphorylation of histone H3 at both serl0 and ser28 is coupled to dynamic acetylation of histone H3 where H3 phosphorylated at serine 28 had a higher steady state of acetylation than that of H3 phosphorylated at serine 10 It was observed that histone H3 phosphorylation at ser28 is regulated by the cell cycle and has been used as a mitotic marker As with phosphorylated ser10 H3 phosphorylation at ser28 also plays an important role in neoplastic cell transformation Several protein kinases including aurora B PPI and PKC are responsible for histone H3 phosphorylation at ser28 Inhibition or activation of these protein kinases can cause a change in intracellular histone H3 phosphorylation at ser28 Detection of the change in histone H3 phosphorylation at ser28 associated with the cell cycle apoptosis and inhibitor or activator treatment would provide useful information for better understanding the pathological processes of certain diseases and for protein kinase targeted drug development The EpiQuik Global Histone H3 Phosphorylation Ser28 Assay Kit Fluorometric provides a tool for measuring global phospho histone H3 ser28 The kit has the following features e Quick and efficient procedure which can be finished within 3 hours e Innovative fluor
9. ound to assay wells Sa Add detection antibody al after wash Add fluoro developing solution for fluorescence development and measurement Schematic Procedure for Using the Global Histone H3 Phosphorylation Ser28 Assay Kit Fluorometric PROTOCOL 1 a Prepare histone extracts from cells tissues treated or untreated by using your own successful method acid extraction or high salt extraction b For your convenience and best results Epigentek offers the EpiQuik Total Histone Extraction Kit Cat OP 0006 optimized for use in the EpiQuik modified histone quantification series c Preparation of histone extracts can also be performed using the attached procedure See Appendix Histone extracts can be used immediately or stored at 80 C for future use 2 Determine the number of strip wells required Leave these strips in the plate frame remaining unused strips can be placed back in the bag Seal the bag tightly and store at 4 C Dilute F1 with distilled water pH 7 2 7 5 at 1 9 ratio 1 ml of F1 9 ml of distilled water 3 Add 50 ul of F2 into each well For the sample add 1 2 ug of the histone extract into the sample wells For the standard curve dilute the standard control with F2 to 1 100 ng ul at 5 7 points e g 1 5 3 6 12 25 50 and 100 ng ul Add 1 ul of standard control at the different concentrations into the standard wells For the blank do not add any nuclear extracts or standard control protein
10. zed mixture to a 15 ml conical tube and centrifuge at 3000 rpm for 5 min at 4 C If total mixture volume is less than 2 ml transfer mixture to a 2 ml vial and centrifuge at 10 000 rpm for 1 min at 4 C Remove supernatant For cells treated and untreated harvest cells and pellet the cells by centrifugation at 1000 rpm for 5 min at 4 C Resuspend cells in TEB buffer at 10 cells ml and lyse cells on ice for 10 min with gentle stirring Centrifuge at 3000 rpm for 5 min at 4 C If total volume is less than 2 ml transfer cell lysates to a 2 ml vial and centrifuge at 10 000 rpm for 1 min at 4 C Remove supernatant 2 Resuspend cell tissue pellet in 3 volumes approx 200 ul 107 cells or 200 mg tissues of extraction buffer 0 5N HCI 10 glycerol and incubate on ice for 30 min 3 Centrifuge at 12 000 rpm for 5 min at 4 C and remove the supernatant fraction to new vial 4 Add 8 volumes approx 0 6 ml 107 cells or 200 mg tissues of acetone and leave at 20 C overnight 5 Centrifuge at 12 000 rpm for 5 min and air dry the pellet Dissolve the pellet in distilled water 30 50 ul 10 cells or 200 mg tissues 6 Quantify the protein concentration Aliquot the extract and store the extract at 20 C or 80 C TROUBLESHOOTING No Signal for Both the Standard Control and the Samples Reagents are added incorrectly Check if reagents are added in order and if some steps of the procedure are omitted by mistake 110 Bi County Blvd
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