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1. T1012 T1019 T1014 T1013 T1020 Microplate assays per kit 600 1800 15 000 Lysis Solution 70 mL 210 mL 1 75 L OR 5X Z Buffer 80 mL 240 mL 2L Galacton Star substrate 1 2 mL 3 6 mL 30 mL Reaction Buffer Diluent 60 mL 180 mL 1 5L 1 Lysis Solution 100 mM potassium phosphate pH 7 8 0 2 Triton X 100 for mammalian cells in T1012 T1014 T1013 OR 5X Z Buffer 0 5 M sodium phosphate pH 7 1 50 mM KCI 5 mM MgSO for yeast cells in T1019 T1020 2 Galacton Star substrate 50X concentrate 3 Reaction Buffer Diluent 100 mM sodium phosphate pH 7 5 1 mM magnesium chloride 5 Sapphire I enhancer lil B GALACTOSIDASE DETECTION PROTOCOL Please read the entire Protocol and Notes sections before proceeding A Preparation of Mammalian Cell Extracts Non adherent cells should be pelleted and rinsed Sufficient Lysis Solution should be added to cover the pellet then the 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 7 AN 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 twice with PBS 3 Add Lysis Solution to cover cells Use 250 ul per 60 mm plate 4 Detach cells from plate with a cell scraper 5 Transfer the cell lysate to a microfuge tube an2. 4 Measure signal in a luminometer for 0 1 1 sec well F Protocol Notes 1 Dithiothreitol DTT may be added to Lysis Solution to 0 5 mM to stabilize B galactosidase activity However DTT may increase assay background and high concentrations will decrease light emission half life If low background and extended half life is critical DTT should be omitted 2 The amount of extract required may vary depending on 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 3 Measurements can be performed after 20 30 min if the time between Reaction Buffer addition and light measurement is the same 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 Galacto Star Lysis Buffer or Z Buffer 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
3. 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 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 17 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 18 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 HID 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 u
4. 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 VAS 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 V REFERENCES 1 Martin CS CEM Olesen B Liu JC Voyta JL Shumway R R Juo and Bronstein 1997 Continuous sensitive detection of B galactosidase with a novel chemiluminescent 1 2 dioxetane In Bioluminescence and Chemiluminescence Molecular Reporting with Photons Hastings JW Kricka LJ and Stanley PE eds p 525 528 John Wiley Chichester England 2 Bronstein J Fortin JC Voyta CEM Olesen and LJ Kricka 1994 Chemiluminescent reporter gene assays for B galactosidase 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 3 Bronstein I CS Martin JJ Fortin CEM Olesen and JC Voyta 1996 Chemiluminescence sensitive detection technology for reporter gene assays Clin Chem 42 9 1542 1546 4 Jain VK and IT Magrath 1991 A chemiluminescent assay for quantitation of galactosidase in the femtogram range Application to quantitatio
5. transgenic mice made with B gal tagged mouse embryonic stem cells 18 19 The system is also formatted for use with yeast cells and is ideally suited for reporter gene assays in yeast 20 or the study of protein protein interactions with the yeast two hybrid system 21 22 Galacton Star substrate has been used for reporter gene assays in bacterial cells with modified lysis reagents 23 Two novel applications demonstrated have been a cell death assay by measurement of B gal released into culture media 24 and a stop codon read through assay using a constitutively expressed f gal luciferase fusion construct 25 The Galacto Star assay system has wide application to cell based assays that use B gal reporter as a read out for gene expression in many cell types and tissues from whole animals or as a functional read out for viral function immune cell activation cell death and mRNA processing Q O 75 Maximum Signal sm Galacto Stas at 25 C Galacto Star at 30 C 90 120 Time min 180 Figure 1 Galacto Star assays were performed with purified B galactosidase 500 pg Repeated measurements were made with the Applied Biosystems TR717 microplate luminometer at either room temperature 25 C or 30 C Data is presented as the percentage of the maximum signal The dotted line indicates 95 of maximum signal Il SYSTEM COMPONENTS Shelf life for all Galacto Star kit components is 1 year at 4 C
6. 05 A simple rapid sensitive method detecting homoserine lactone HSL related compounds in microbial extracts J Microbiol Methods 65 1 32 37 Schotte P G Denecker A Van Den Broeke P Vandenabeele GR Cornelis and R Beyaert 2004 Targeting Rac1 by the Yersinia effector protein YopE inhibits caspase 1 mediated maturation and release of interleukin 1B J Biol Chem 279 24 25134 25142 Carnes J M Jacobson L Leinwand and M Yarus 2003 Stop codon suppression via inhibition of eRF1 expression RNA 9 648 653 For complete reference list please see http www appliedbiosystems com Product amp Service Literature 13
7. 1 Fan S M Gao Q Meng JJ Laterra MH Symons S Coniglio RG Pestell ID Goldberg and EM Rosen 2005 Role of NF B signaling in hepatocyte growth factor scatter factor mediated cell protection Oncogene 24 1749 1766 12 Silver SJ EL Davies L Doyon and Rebay 2003 Fucntional dissection of eyes absent reveals new modes of regulation within the retinal determination gene network Mol Cell Biol 23 17 5989 5999 13 Simmons G AJ Rennekamp N Chai LH Vandenberghe JL Riley and P Bates 2003 Folate receptor alpha and caveolae are not required for Ebola virus glycoprotein mediated viral infection J Virol 77 24 13433 13438 14 Fouts T K Godfrey K Bobb D Montefiori CV Hanson VS Kalyanaraman A DeVico and R Pal 2002 Crosslinked HIV 1 envelope CD4 receptor complexes elicit broadly cross reactive neutralizing antibodies in rhesus macaques Proc Natl Acad Sci USA 99 18 11842 11847 12 15 16 17 18 19 20 21 22 23 24 25 Kensinger RD BJ Catalone FC Krebs B Wigdahl and C L Schengrund 2004 Novel polysulfated galactose derivatized dendrimers as binding antagonists of Human Immunodeficiency Virus Type 1 infection Antimicrob Agents Chemotherapy 48 5 1614 1623 Keeton EK and M Brown 2005 Cell cycle progression stimulated by tamoxifen bound estrogen receptor o and promoter specific effects in breast cancer cells deficient in N CoR and SMRT Mol Endocrinol 19 6 1543 1554 Serna A MC
8. KS App ied ns Applied Biosystems 35 Wiggins Avenue Bedford MA 01730 800 542 2369 781 271 0045 Press 2 Galacto Star System Chemiluminescent Reporter Gene Assay System for the Detection of B Galactosidase P N 11012 T1013 T1014 T1019 T1020 Contents Page PREFACE sonion AA Aa Aviat nea 1 l INTRODUCTION narica nea Ea raaa KAE EENE EA ENEE NEEE RAEE E NNE 2 Il SYSTEM COMPONENT Grecie in AEE EER 3 Ill B GALACTOSIDASE DETECTION PROTOCOOL 0 cc ccecssssteeeeeesees 4 A Preparation of Mammalian Cell Extracts cccccceeeeeeeeeeeeeees 4 B Preparation of Yeast Cell Extracts ccccecceceeeeeeeeeeeeteeeseneees 4 C Direct Lysis Protocol for Microplate Cultures ecsee 5 D Detection Protocol for Tube Luminomete s 0 eeeeeeeeeees 5 E Detection Protocol for Microplate Luminometers 000 6 F Protocol NOteS siasi a a a a a T A 6 IV APPENDICES tiiccvcscticocathencsvevansscvauieasd ARENAN REENA NAAA ERAEN CASERA 6 A Preparation of CONTOS iesma a a a a 6 B Inactivation of Endogenous B Galactosidase ccccccceeceeeeesteeeteees 7 C Use of Luminometers cececec ec eee cece cece ee eeeeeeeeeeeeaeeeeeeeeeeas 7 D Safety rnea a Widens teies cada ete sk bee acme tee ees 7 V REFERENCES vassk lt cecsaethevecvedsatacecegenscduecsyatetecesssedtsevecyied ence cPust ARARE EEA 12 Part Number T9017 Revision D Rev
9. MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal 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 transport 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 Inthe 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 e Open To view the document Print Target To print the document e 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 CHEMICAL WASTE SAFETY Chemica
10. Ramirez A Soukhanova and LJ Sigal 2003 Cutting edge Efficient MHC class cross presentation during early vaccinia infection requires the transfer of proteasomal intermediates between antigen donor and presenting cells J mmunol 171 5668 5672 Rokosh DG and PC Simpson 2002 Knockout of the 1A C adrenergic receptor subtype The amp 1A C is expressed in resistance arteries and is required to maintain arterial blood pressure Proc Natl Acad Sci USA 99 14 9474 9479 Farhadi HF P Lepage R Forghani HCH Friedman W Orfali L Jasmin W Miller TJ Hudson and AC Peterson 2003 A combinatorial network of evolutionarily conserved Myelin Basic Protein regulatory sequences confers distinct glial specific phenotypes J Neurosci 23 32 10214 10223 Olesnicky NS AJ Brown SJ Dowell and LA Casselton 1999 A constitutively active G protein coupled receptor causes mating self compatibility in the mushroom Coprinus EMBO J 18 2756 2763 Daub M J Jockel T Quack CK Weber F Schmitz UR Rapp A Wittinghofer and C Block 1998 The RafC1 cysteine rich domain contains multiple distinct regulatory epitopes which control Ras dependent Raf activation Mol Cell Biol 18 6698 6710 Scharf KD H Heider Hohfeld R Lyck E Schmidt and L Nover 1998 The tomato Hsf system HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules Mol Cell Biol 18 4 2240 2251 Singh MP and M Greenstein 20
11. agent injection A direct lysis protocol for measurement of B galactosidase activity in microplate cell cultures is also provided The bacterial B galactosidase gene is widely used as a reporter enzyme for the study of gene regulation Tropix chemiluminescent 1 2 dioxetane substrates for B galactosidase including Galacton product discontinued Galacton Plus and Galacton Star provide highly sensitive enzyme detection 1 4 and have been utilized in reporter assays in both mammalian cell culture and tissue extracts and Galacton Plus substrate is incorporated in a combined assay for firefly luciferase and B galactosidase activities in cell extracts 5 6 Dual Light assay system P N T1000 Chemiluminescent reporter assays may be conducted in cells or tissues that have endogenous f galactosidase Reduction of endogenous activity may be achieved using heat inactivation 7 Tissue extracts may require the use of protease inhibitors 8 Applications The Galacto Star assay system is used widely for traditional reporter gene assays in transfected mammalian cells 9 11 and in insect cells 12 A wide variety of applications have been performed including viral function assays with B Gal encoding pseudovirions 13 and MAGI cells 14 15 normalization of siRNA transfection 16 and as a reporter read out for epitope recognition by an engineered CTL hybridoma cell line 17 The Galacto Star system has been used to assay tissues extracts of
12. d centrifuge for 2 min to pellet debris 6 Transfer extract supernatant to a fresh tube Use immediately or store at 70 C B Preparation of Yeast Cell Extracts 1 Dilute 5X Z Buffer 1 5 in H20 Add DTT to 0 5 mM if desired see Note 1 2 Transfer 1 5 mL of yeast culture ODsoo 0 5 to a microfuge tube Centrifuge at 12 000g for 30 sec If ODgoo is lt 0 4 use gt 1 5 mL 3 Remove supernatant and resuspend cell pellet in 1 5 mL of 1X Z Buffer 4 Centrifuge at 12 000g for 30 sec 5 Remove supernatant and resuspend pellet in 300 ul of 1X Z Buffer 6 Transfer 100 ul to a fresh tube Store the remainder at 4 C 7 Perform 2 freeze thaw cycles Freeze in liquid No for 60 sec Thaw cells at 37 C for 60 sec 8 Centrifuge at 12 000g for 5 min at 4 C 9 Transfer supernatant to a fresh tube Use immediately or store at 70 C C Direct Lysis Protocol for Microplate Cultures This procedure is designed for adherent cels growing in 96 well tissue culture treated luminometer plates Perform assays in triplicate at room temperature Heat inactivation of endogenous f galactosidase activity is not effective with this protocol For the following hazards see the complete safety alert descriptions in Appendix D Safety on page 7 A 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 cells once with PBS 3 Add 10 ul of Lysi
13. d 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 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 location
14. 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 national 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 10 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 i
15. 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 J 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 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 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
16. ision 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 INCLUDING 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 Star System Trademarks Applied Biosystems AB Design Dual Light Galacton Galacton Plus Galacton Star and Tropix are registered trademarks and Galacto Star and Sapphire II are trademarks of Applied Biosystems Inc or its subsidiaries in the US and or 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 hazar
17. l waste hazards AN CAUTION HAZARDOUS WASTE Refer to Material Safety Data Sheets and local regulations for handling and disposal A WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death A 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 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
18. n of B galactosidase in lacZ transfected cells Anal Biochem 199 119 124 5 Bronstein CS Martin CEM Olesen and JC Voyta 1997 Combined luminescent assays for multiple enzymes p 451 457 n Bioluminescence and Chemiluminescence Molecular Reporting with Photons Hastings J W Kricka L J and Stanley P E eds John Wiley Chichester England 6 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 7 Young DC SD Kingsley KA Ryan and FJ Dutko 1993 Selective inactivation of eukaryotic p galactosidase in assays for inhibitors of HIV 1 TAT using bacterial B galactosidase as a reporter enzyme Anal Biochem 215 24 30 8 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 9 De Bosscher K CS Hill and FJ Nicolas 2004 Molecular and functional consequences of Smad4 C terminal missense mutations in colorectal tumour cells Biochem J 379 209 216 10 Pittler SJ Y Zhang S Chen AJ Mears DJ Zack Z Ren PK Swain S Yao A Swaroop and JB White 2004 Functional analysis of the rod photoreceptor cGMP phosphodiesterase o subunit gene promoter J Biol Chem 279 19 19800 19807 1
19. s 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 INTRODUCTION The Tropix Galacto Star chemiluminescent reporter assay system is designed for rapid and sensitive detection of B galactosidase activity in cell lysates The assay incorporates Tropix Galacton Star substrate for B galactosidase with Tropix Sapphire II luminescence enhancer to produce glow light emission kinetics The Galacto Star assay has a wide dynamic range enabling detection from 2 fg to 20 ng of purified B galactosidase 1 The system is ideally suited for use with either mammalian or yeast cells The Galacto Star assay provides a simplified method for detecting B galactosidase Cell lysate is incubated with Reaction Buffer containing Galacton Star and Sapphire Il enhancer until maximum light emission is reached typically 30 90 min depending upon assay temperature Fig 1 after peak light emission remains constant for nearly 1 hour Light signal is measured in a luminometer without the need for re
20. s Solution to each well and incubate for 10 min 4 Continue with the procedure for Detection with Microplate Luminometers Section E omitting Step 2 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 7 AN WARNING CHEMICAL HAZARDS Reaction Buffer Diluent 1 Dilute Galacton Star substrate 1 50 with Reaction Buffer Diluent to make Reaction Buffer Prepare only enough for daily use 300 pL tube Equilibrate to room temperature 2 Transfer 2 20 uL of extract to luminometer tubes see Note 2 3 Add 300 ul of Reaction Buffer mix and incubate for 30 90 min until light emission is maximal see Figure 1 and Note 3 4 Measure signal in a luminometer for 0 1 1 sec tube E 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 7 J WARNING CHEMICAL HAZARDS Reaction Buffer Diluent 1 Dilute Galacton Star substrate 1 50 with Reaction Buffer Diluent to make Reaction Buffer Prepare only enough for daily use 200 uL well Equilibrate to room temperature 2 Transfer 2 10 uL of extract to microplate wells see Note 2 3 Add 100 uL of Reaction Buffer mix and incubate for 30 90 min until light emission is maximal see Figure 1 and Note 3
21. s 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 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 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 11 Specific chemical alerts AN WARNING CHEMICAL HAZARD Lysis Solution causes eye skin and respiratory
22. sing 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 detector 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 GENERAL CHEMICAL SAFETY Chemical hazard warning 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 AN 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
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