Galacto-Light™ Plus System Protocol (PN T9003J)
Contents
1. product discontinued chemiluminescent reporter assay systems are designed for rapid and sensitive detection of B galactosidase reporter enzyme in cell lysates The Galacto Light Plus reporter assay incorporates Galacton Plus chemiluminescent substrate for B galactosidase with Tropix Sapphire lI luminescence enhancers The assay has a wide dynamic range enabling detection from 2 fg to 20 ng of purified B galactosidase 1 3 B galactosidase detection is simple and fast Cell lysate is incubated with reaction buffer for 15 minutes to 1 hour Galacton Plus substrate in the reaction buffer is cleaved by the enzyme The sample is then placed in a luminometer and Accelerator Il is added terminating enzyme activity and triggering light emission from the cleaved substrate Galacton Plus chemiluminescent substrate emits light at a near constant level with a half life of approximately 180 minutes after the addition of Accelerator Il This substrate is ideal for use with instruments without reagent injectors The Lysis Solution provided may be replaced with alternative lysis solutions and lysis procedures if co transfected reporters require different conditions Alternative procedures should be compared with the Galacto Light Plus lysis protocol to ensure optimal assay performance The bacterial B galactosidase gene is widely used as a reporter enzyme for the study of gene regulation and more recently in systems for identification of protein protein intera2. CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations MSDSs The MSDSs for any chemicals supplied by Applied Biosystems are available to you free 24 hours a day For instructions on obtaining MSDSs see MSDSs on page 8 IMPORTANT For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer How to Obtain Support For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems web site you can e Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents e Download PDF documents e Obtain information about customer training e Download software updates and patches l INTRODUCTION The Tropix Galacto Light Plus and Galacto Light
3. Product amp Service Literature 12
4. viral function assays with B Gal encoding MAGI cells 22 and targeted gene expression for gene therapy 23 A novel B galactosidase peptide complementation system utilizing Galacton Plus substrate has been used in mammalian cells to detect intracellular protein protein interactions 24 25 In addition B galactosidase peptide complementation has been used in an assay for myoblast cell fusion in cell culture 24 26 Cytotoxicity has been monitored by measurement of B gal reporter enzyme released into culture media 27 and an MRNA trans splicing assay in primary fetal fibroblasts was performed by measuring reconstitution of B Gal activity from partial RNA transcripts 28 The Galacto Light Plus assay system has wide application to assays that use the B gal reporter as a functional read out enabling highly sensitive detection in many different types of cells and organisms Il SYSTEM COMPONENTS Galacto Light Plus kits P Ns T1007 T1011 T1009 utilize Galacton Plus substrate and Accelerator Il Shelf life for all kits is 1 yr at 4 C T1007 T1011 T1009 Microplate assays per kit 600 1800 15 000 Lysis Solution 70 mL 210 mL 1 75 L Substrate Solution 0 4 mL 1 2 mL 10 mL Reaction Buffer Diluent 40 mL 120 mL 1L Accelerator 70 mL 210 mL 1 75 L 1 Lysis Solution 100 mM potassium phosphate pH 7 8 0 2 Triton X 100 2 Chemiluminescent Substrate Galacton Plus 100X concentrate 3 Reaction Buffe
5. Applied Biosystems 35 Wiggins Avenue Bedford MA 01730 800 542 2369 781 271 0045 Press 2 http www appliedbiosystems com Applied KS Biosystems Galacto Light Plus System Chemiluminescent Reporter Gene Assay System for the Detection of B Galactosidase P N T1007 T1011 T1009 Contents PREFACE INTRODUCTION SYSTEM COMPONENTS B GALACTOSIDASE DETECTION PROTOCOL Preparation of Cell Extracts from Cultured Cells Direct Lysis Protocol for Microplate Cultures Detection with Microplate Luminometers Detection with Tube Luminometers Protocol Notes PPENDICES Preparation of Controls Inactivation of Endogenous Galactosidase Use of Luminometers Safety EFERENCES gt B C D E A A B C D R g o FPNODNDOOUAAARWWWHNDM Part Number T9003 Revision J Revision Date October 2008 For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLU
6. B Inactivation of Endogenous B Galactosidase Some cell lines exhibit endogenous B galactosidase activity This may lead to high background which will decrease the sensitivity of the assay A procedure for heat inactivation of endogenous B galactosidase activity has been described 5 A modified version of this protocol has also been described for use with tissue extracts in which protease inhibitors are used in conjunction with the heat inactivation procedure 6 These procedures should be performed prior to the Detection Protocol Inactivation of B Galactosidase Activity 1 Heat the extract for 50 60 min at 48 C 2 Proceed with detection Section IIIC or IHD NOTE PMSF to 0 2 mM and leupeptin to 5 ug mL may be added to Lysis Solution just before use if protease activity may be present NOTE AEBSF a water soluble analog of PMSF Sigma A 8456 may replace of PMSF Sigma P 7626 Leupeptin Sigma L 2884 is recommended C Use of Luminometers We recommend using a single mode luminometer or a multi mode detection insturment set for luminescence measurement to measure the light emission from 96 or 384 well microplates The linear range of detection will vary according to cell type and on the reporter enzyme expression level The number of cells or sample volume used per well should be optimized to prevent a measurement signal that is outside the linear range of the luminometer Extremely high light signals can saturate the detecto
7. D Safety on page T N WARNING CHEMICAL HAZARDS Lysis Solution 1 Add DTT to 0 5 mM to the required volume of Lysis Solution if desired see Note 1 2 Rinse cell cultures once with PBS 3 Add 10 uL of Lysis Solution to each well and incubate for 10 min 4 Continue with the procedure for Detection with Microplate Luminometers Section C omitting Step 3 C Detection with Microplate Luminometers Perform assays in triplicate at room temperature For the following hazards see the complete safety alert descriptions in Appendix D Safety on page T IK WARNING CHEMICAL HAZARDS Galacton Plus substrate Lysis Solution Reaction Buffer Diluent Accelerator ll 1 Dilute Galacton Plus substrate 1 100 with Reaction Buffer Diluent to make Reaction Buffer Prepare only enough for one day s use 70 uL well 2 Equilibrate Reaction Buffer and Accelerator I to room temperature 3 Transfer 2 20 uL of extract to microplate wells see Note 2 4 Add 70 ul of Reaction Buffer per well and incubate for 30 60 min see Note 3 5 Place plate in luminometer Inject 100 ul of Accelerator ll After a 1 2 sec delay read signal for 0 1 1 sec well D Detection with Tube Luminometers Perform assays in triplicate at room temperature For the following hazards see the complete safety alert descriptions in Appendix D Safety on page Ti AN WARNING CHEMICAL HAZARDS Galacton Plus substrate Reaction Buffer Diluent Ac
8. DING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES Literature Citation When describing a procedure for publication using this product please refer to it as the Galacto Light Plus System Trademarks Applied Biosystems AB Design Dual Light Galacton Galacton Plus Galacton Star and Tropix are registered trademarks and Galacto Light Galacto Light Plus Galacto Star and Sapphire II are trademarks of Applied Biosystems Inc or its subsidiaries in the US and certain other countries All other trademarks are the sole property of their respective owners Copyright 2008 Applied Biosystems All rights reserved PREFACE Safety Information Note For general safety information see this Preface and Appendix D Safety on page 7 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Safety Alert Words Four safety alert words appear in Applied Biosystems user documentation at point in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical
9. U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials IMPORTANT Additional information about biohazard guidelines is available at www cdc gov 5 CHEMICAL ALERTS For the definitions of the alert words IMPORTANT CAUTION WARNING and DANGER see Safety alert words on page 1 General alerts for all chemicals EXAMPLE Avoid contact with skin eyes and or clothing Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 10 Specific chemical alerts VAN WARNING CHEMICAL HAZARD Accelerator causes skin and respiratory tract irritation and causes eye burns Harmful if swallowed or absorbed through skin May cause allergic reaction Avoid breathing vapor Use with adequate ventilation Avoid contact with eyes and skin Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves J WARNING CHEMICAL HAZARD Accelerator Il causes skin and respiratory tract irritation V N Pee ee ie and causes eye burns Harmful if swallowed
10. aste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical waste safety guidelines To minimize the hazards of chemical waste Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or nationa
11. celerator ll 1 Dilute Galacton Plus substrate 1 100 with Reaction Buffer Diluent to make Reaction Buffer Prepare only enough for one day s use 200 uL tube 2 Equilibrate Reaction Buffer and Accelerator Il to room temperature 3 Transfer 2 20 uL of extract to luminometer tubes see Note 2 4 Add 200 ul of Reaction Buffer per tube and incubate for 30 60 min see Note 3 5 Place tube in luminometer Inject 300 ul of Accelerator ll After a 1 2 sec delay read signal for 0 1 1 sec tube E Protocol Notes 1 Dithiothreitol DTT not included may be added to Lysis Solution to 0 5 mM to stabilize B galactosidase activity However DTT may increase assay background and will decrease the half life of light emission of Galacton Plus If low background and extended half life of light emission is critical for instance if manual addition of Accelerator Il is being used DTT should be omitted If DTT must be used adding H20 to Accelerator Il to 10 mM add 1 uL of 30 H20 per 1 mL of Accelerator I will prevent rapid decay of signal half life 2 The amount of cell extract required depends upon the B galactosidase expression level Use 2 5 ul or 10 20 ul of extract for samples with high or low levels of enzyme respectively For reproducible results assay the same volume of sample every time Lysis Solution may be added to equalize sample volumes 3 Due to the light emission kinetics of the reaction it is important that each well
12. ctions 1 2 Dioxetane substrates for B galactosidase including Galacton product discontinued Galacton Plus and Galacton Star Tropix Galacto Star system provide highly sensitive enzyme detection and have been used widely in reporter assays in both cell and tissue extracts The Galacton Plus substrate is also incorporated into a combined assay for dual detection of firefly luciferase and B galactosidase activities in the same cell extract sample Tropix Dual Light system 4 Chemiluminescent B galactosidase reporter assays may be conducted in cells or tissues that have endogenous enzyme activity Endogenous activity can be significantly reduced by heat inactivation 5 Tissue extracts may require the use of protease inhibitors 6 Applications The Galacto Light Plus and Galacto Light assay systems are widely used for traditional reporter gene assays in transfected mammalian cell lines in culture 7 9 and have been used with primary culture cells 10 tissue extracts from transgenic mice 11 12 frog embryo extracts 13 Drosophila embryo extracts 14 protozoan parasites 15 and in Pseudomonas bacterial cells 16 These assay systems have also been utilized in yeast reporter gene assays 17 18 and for the study of protein protein interactions with the yeast two hybrid system 19 20 and DNA protein interactions with the one hybrid system 21 A variety of applications have been performed with mammalian cells such as
13. e and contributes to the formation of tracheal and nervous systems Development 130 719 728 Beetham JK KS Myung JJ McCoy ME Wilson and JE Donelson 1997 Glycoprotein 46 mRNA abundance is post transcriptionally regulated during development of Leishmania chagasi promastigotes to an infectious form J Biol Chem 272 28 17360 17366 Yarwood JM EM Volper and EP Greenberg 2005 Delays in Pseudomonas aeruginosa quorum controlled gene expression are conditional Proc Natl Acad Sci USA 102 25 9008 9013 Remacle JE G Albrecht R Brys GH Braus and D Huylebroeck 1997 Three classes of mammalian transcription activation domain stimulate transcription in Schizosaccharomyces pombe EMBO J 16 18 5722 5729 Stoldt VR A Sonneborn CE Leuker and JF Ernst 1997 Efg1p an essential regulator of morphogenesis of the human pathogen Candida albicans is a member of a conserved class of bHLH proteins regulating morphogenetic processes of fungi EMBO J 16 18 1982 1991 Bourne Y MH Watson MJ Hickey W Holmes W Rocque SI Reed and JA Tainer 1996 Crystal structure and mutational analysis of the human CDK2 kinase complex with cell cycle regulatory protein CksHs1 Cell 84 863 874 Schumacher S K Laass S Kant Y Shi A Visel AD Gruber A Kotlyarov and M Gaestel 2004 Scaffolding by ERK3 regulates MK5 in development EMBO J 23 4770 4779 Wolf SS K Roder and M Schweizer 1996 Construction of a reporter plasmid that allows expression libraries t
14. have an identical incubation time prior to measurement Reaction Buffer should be added to samples in the same sequence that they will be measured and the timing should correspond to the timing of Accelerator II addition measurement for all samples IV APPENDICES A Preparation of Controls Positive Control Reconstitute lyophilized B galactosidase Sigma G 5635 to 1 mg mL in 0 1 M sodium phosphate pH 7 0 0 1 BSA Store at 4 C Generate a standard curve by serially diluting in Lysis Solution containing 0 1 BSA 2 20 ng of enzyme should be used as an upper detection limit Purified enzyme provides a positive control for the assay reagents as well as a means to determine the range of detection of the luminometer instrumentation if desired The purified enzyme standard curve is not intended or accurate for absolute quantitation of reporter enzyme concentrations as the specific activity of the purified enzyme preparation and the reporter enzyme may differ significantly Additional positive controls can include use of control B galactosidase constructs that provide constitutive expression of reporter enzyme as a positive control for cell transfection Negative Control Assay a volume of mock transfected extract equivalent to that of experimental extract to determine endogenous cellular background In experiments involving induction of reporter expression uninduced cells should be assayed as a negative control for total assay background
15. l environmental and health regulations Waste disposal If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply 4 BIOLOGICAL HAZARD SAFETY General biohazard A WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following
16. n and LJ Kricka 1994 Chemiluminescent reporter gene assays for B galactosidase B glucuronidase and secreted alkaline phosphatase p 20 23 In Bioluminescence and Chemiluminescence Fundamentals and Applied Aspects Campbell AK Kricka LJ and Stanley PE eds John Wiley Chichester England Bronstein CS Martin JJ Fortin CEM Olesen and JC Voyta 1996 Chemiluminescence sensitive detection technology for reporter gene assays Clin Chem 42 9 1542 1546 Martin CS PA Wight A Dobretsova and Bronstein 1996 Dual luminescence based reporter gene assay for luciferase and B galactosidase BioTechniques 21 3 520 524 Young DC SD Kingsley KA Ryan and FJ Dutko 1993 Selective inactivation of eukaryotic B galactosidase in assays for inhibitors of HIV 1 TAT using bacterial B galactosidase as a reporter enzyme Anal Biochem 215 24 30 Shaper N A Harduin Lepers and JH Shaper 1994 Male germ cell expression of murine B4 galactosyltransferase A 796 base pair genomic region containing two cAMP responsive element CRE like elements mediates male germ cell specific expression in transgenic mice J Biol Chem 269 25165 25171 11 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Graslund T X Li L Magnenat M Popkov and CF Barbas III 2005 Exploring strategies for the design of artificial transcription factors J Biol Chem 280 5 3707 3714 Yeaman
17. ner with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical safety guidelines To minimize the hazards of chemicals Read and understand the Material Safety Data Sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page 8 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal 2 MSDSs About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle tra
18. nsport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining MSDSs The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day To obtain MSDSs 1 Go to www appliedbiosystems com click Support then select MSDS 2 In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer 3 CHEMICAL WASTE SAFETY Chemical waste hazards CAUTION HAZARDOUS WASTE Refer to Material Safety Data Sheets and local regulations for handling and disposal AN WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each w
19. o be exploited for the one hybrid assay system BioTechniques 20 4 568 573 Neurath AR N Strick and Y Y Li 2002 Anti HIV 1 activity of anionic polymers a comparative study of candidate microbicides BMC Infectious Diseases 2 27 www biomedcentral com 1471 2334 2 27 Ozturk Winder F M Renner D Klein M Muller B Salmons and WH Gunzburg 2002 The murine whey acidic protein promoter directs expression to human mammary tumors after retroviral transduction Cancer Gene Ther 9 421 431 Mohler WA and HM Blau 1996 Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells Proc Natl Acad Sci USA 93 12423 12427 Rossi F CA Charlton and HM Blau 1997 Monitoring protein protein interactions in intact eukaryotic cells by B galactosidase complementation Proc Natl Acad Sci USA 94 8405 8410 Charlton CA WA Mohler GL Radice RO Hynes and HM Blau 1997 Fusion competence of myoblasts rendered genetically null for N cadherin in culture J Cell Biol 138 331 336 Schafer H A Schafer AF Kiderlen KN Masihi and R Burger 1997 A highly sensitive cytotoxicity assay based on the release of reporter enzymes from stably transfected cell lines J mmunol Methods 204 89 98 Duan D Y Yue and JF Engelhardt 2001 Expanding AAV packaging capacity with trans splicing or overlapping vectors A quantitative comparison Mol Therapy 4 4 383 391 For complete updated reference list please see http Awww appliedbiosystems com
20. or absorbed through skin May cause allergic reaction Avoid breathing vapor Use with adequate ventilation Avoid contact with eyes and skin Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves WARNING CHEMICAL HAZARD Galacton Plus substrate may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves WARNING CHEMICAL HAZARD Lysis Solution causes eye skin and respiratory tract irritation Avoid breathing vapor Use with adequate ventilation Avoid contact with eyes and skin Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves WARNING CHEMICAL HAZARD Reaction Buffer Diluent may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves WARNING CHEMICAL HAZARD Substrate Solution may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves REFERENCES Jain VK and IT Magrath 1991 A chemiluminescent assay for quantitation of galactosidase in the femtogram range Application to quantitation of B galactosidase in lacZ transfected cells Anal Biochem 199 119 124 Bronstein J Fortin JC Voyta CEM Olese
21. r very unlikely for experimental samples resulting in erroneous measurements Refer to your luminometer user s manual and use the positive control serial dilution curve to determine the upper limit for your specific luminometer Contact Applied Biosystems Technical Support for additional questions D Safety 1 GENERAL CHEMICAL SAFETY Chemical hazard warning AN AN A AN WARNING CHEMICAL HAZARD Before handling any chemicals refer to the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety contai
22. r Diluent 100 mM sodium phosphate pH 8 0 1 mM MgClo 4 Accelerator Il Ready to Use reagent containing Sapphire II enhancer III B GALACTOSIDASE DETECTION PROTOCOL Please read the entire Protocol and Notes sections before proceeding A Preparation of Extracts from Cultured Cells Non adherent cells should be pelleted and rinsed Sufficient Lysis Solution should be added to cover the pellet then cells should be resuspended by pipetting Continue at step 5 below For the following hazards see the complete safety alert descriptions in Appendix D Safety on page T A N WARNING CHEMICAL HAZARDS Lysis Solution 1 2 Add DTT to 0 5 mM to the required volume of Lysis Solution if desired see Note 1 Rinse cell cultures twice with PBS Add Lysis Solution to cover cells Use 250 ul per 60 mm plate Detach cells from plate with a cell scraper Transfer the cell lysate to a microfuge tube and centrifuge for 2 min to pellet debris Transfer extracts Supernatant to a fresh tube Use immediately or store at 70 C B Direct Lysis Protocol for Microplate Cultures This procedure is designed for adherent cells growing in 96 well tissue culture treated luminometer plates Perform assays in triplicate at room temperature Heat inactivation of endogenous galactosidase activity has not been found to be effective with this protocol For the following hazards see the complete safety alert descriptions in Appendix
23. s C D Wang P P BE Torbett DG Tenen and AD Friedman 2007 C EBPa binds and activates the PU 1 distal enhancer to induce monocyte lineage commitment Hematopoiesis 110 9 3136 3142 Edenberg HJ J Wang H Tian S Pochareddy X Xuei L Wetherill A Goate T Hinrichs S Kuperman Jl Nurnberger Jr M Schuckit JA Tischfield and T Foroud 2008 A regulatory variation in OPRK7 the gene encoding the x opioid receptor is associated with alcohol dependence Hum Mol Gen 17 12 1783 1789 Saller RM F Ozturk B Salmons and WH Gunzburg 1998 Construction and characterization of a hybrid mouse mammary tumor virus murine leukemia virus based retroviral vector J Virol 72 2 1699 1703 Ryan AJ K Fisher CP Thomas and RK Mallampalli 2004 Transcriptional repression of the CTP phosphocholine cytidyltransferase gene by sphingosine Biochem J 382 741 750 Xu L L Renaud JG Mller CF Baicu DD Bonnema H Zhou CS Kappler SW Kubalak MR Zile SJ Conway and DR Menick 2006 Regulation of Ncx1 expression Identification of regulatory elements mediating cardiac specific expression and up regulation J Biol Chem 281 45 34430 34440 Gove C M Walmsley S Nijjar D Bertwistle M Guille G Partington A Bomford and R Patient 1997 Over expression of GATA 6 in Xenopus embryos blocks differentiation of heart precursors EMBO J 16 2 355 368 Liu Q X M Jindra H Ueda Y Hiromi and S Hirose 2003 Drosophila MBF 1 is a co activator for Tracheae Defectiv
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