TnT® Quick Coupled Transcription/Translation Systems Technical
Contents
1. 30 reactions L4540 Bulk Flexi Rabbit Reticulocyte Lysate is available from Promega Wheat Germ Extract Product Size Cat Wheat Germ Extract 30 reactions L4380 Rabbit Reticulocyte Lysate Wheat Germ Extract Combination System Product Size Cat Rabbit Reticulocyte Wheat Germ Extract Combination System 12 reactions L4330 E coliS30 Extract Systems Product Size Cat E coli S30 Extract for Linear Templates a 30 reactions L1030 E coli S30 Extract for Circular DNA 2 30 reactions L1020 E coli T7 S30 Extract System for Circular DNA 30 reactions L1130 Transcend Non Radioactive Translation Detection Systems Product Size Cat Transcend Colorimetric Translation Detection System 30 reactions L5070 Transcend Chemiluminescent Non Radioactive Translation Detection System Colorimetric 30 reactions L5080 Transcend Biotinylated tRNA 30ul L5061 For Laboratory Use Fluorofect Green ys in vitro Translation Labeling System Product Size Cat FluoroTect Green ys in vitro Translation Labeling System 40 reactions L5001 For Laboratory Use Canine Pancreatic Microsomal Membranes Product Size Cat Canine Pancreatic Microsomal Membranes e 50ul Y4041 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Printed in USA Page 24 Revised 9 02 U S Pat Nos 5 283 179 5 641 642. Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 9 02 Page 3 Gene 1 GST Gene 2 Expressed GST pull down TnT System GST Protein 2 ME ii 1 M W Wash E Eluate zk M Marker Autoradiography Western Figure 2 The study of protein protein interactions using the TNT Systems 6 This schematic shows translation of one protein with radioactive S5 gt S methionine in a TNT System reaction Large amounts of the suspected partner protein are expressed and purified A fusion tag most commonly GST is incorporated into this second protein to facilitate purification and subsequent capture steps After the GST fusion protein is immobilized on sepharose GST pulldowns it is mixed with the protein produced in the TNT reaction The sepharose is washed to remove unbound protein and the remaining bound proteins are eluted and analyzed on a gel This technique allows measurement of the protein protein interactions for both pro teins and is often used to verify the in vivo results obtained from yeast two hybrid experiments 2598MA03_9A ll Product Components Product Cat TNTE T7 Quick Coupled Transcription Translation System L1170 TNT SP6 Quick Coupled Transcription Translation System L2080 For Laboratory Use Each system contains sufficient reagents to perform approximately 40 x 50ul translation reac tions In
3. TNT Quick Coupled Transcription Translation Systems Technical Manual No 045 INSTRUCTIONS FOR USE OF PRODUCTS L1170 L1171 L2080 AND L2081 PLEASE DISCARD PREVIOUS VERSIONS All technical literature is available on the Internet at www promega com Please visit the web site to verify that you are using the most current version of this Technical Manual L DESCHID eosa EE E E E EEN 2 Il Product Components cccccccccceeccecececaeeeeeeeeeeaeeeseceeeseseeeeeeessssaeeeeeeeessaaages 4 Ill General Considerations ccc cccccccceeseeeeeeeeeceeeeeeeceeeeseeeeeeseeeeessaeaeeeeeaaes 5 A DNA Template Considerations ccccccccsssseccececeeeseeeeeeeseeeseeeeeessaaeeeeeeeeaeaees 5 B Creating a Ribonuclease Free Environment ccccccceccecsseeeeeeeeeeeeeeeseeeeeeens 6 G WGC Or LS Ce ee oo sas cs oc EEEE E ON 6 IV Translation Procedure ccccccccccccceeceeceeeeeceeeeeeseeeeeeeeeceseeeeesseeeeseeeesseeeeas 6 A General Protocol for TNT Quick Coupled Transcription Translation Reactions Using Plasmid DNA cccceeeeeeeees 7 B General Protocol for TNT T7 Quick Coupled Transcription Translation Reactions Using PCR Generated DNA 8 CG NOIE parece eateries es weet a got pen earntrnte natnde ed E 9 V Positive Control Translation Reactions Using Luciferase 0 10 A Radioactive Luciferase Control Reaction ccc
4. Precast polyacrylamide gels are available from a number of manufacturers For protein analysis NOVEX and Bio Rad Laboratories Inc offer a variety of precast mini gels which are compatible with their vertical electrophoresis and blotter systems These companies offer Tris Glycine Tricine and Bis Tris gels for resolution of proteins under different conditions and over a broad spectrum of protein sizes The NOVEX 4 20 Tris Glycine gradient gels NOVEX Cat Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Page 12 Printed in USA Revised 9 02 EC6025 or EC60355 and the Bio Rad Ready Gel 4 20 Tris Glycine Gel 10 well Bio Rad Cat 161 0903 are convenient for resolving proteins over a wide range of molecular weights In addition to convenience and safety precast gels provide consistent results 1 Once the 50ul translation reaction is complete or at any desired timepoint remove a 1 5ul aliquot and add it to 20ul of SDS sample buffer The remainder of the reaction may be stored at 20 C or at 70 C for long term storage 2 Cap the tube and heat at 100 C for 2 minutes to denature the proteins This may cause protein aggregation A 70 C incubation for 10 minutes may be more appropriate for some samples 3 A small aliquot 5 10ul of the denatured sample can then be loaded
5. the expiration date Ethanol or salt present in Ethanol or salt may inhibit the translation reaction translation Low translation efficiency Certain gene constructs Add 1 3ul of the T7 TNT PCR may require different Mg2 Enhancer and K concentrations for optimal expression Calcium is present inthe Avoid adding calcium to the trans translation reaction lation reaction Calcium may reac tivate the micrococcal nuclease used used to destroy endogenous mRNA in the lysate and result in degrada tion of the DNA or mRNA template Ethanol present in the Residual ethanol should be re translation reaction moved from template DNA preparations and amino acids before they are added to the translation reaction Incubation of the reaction Incubate the reaction at 30 C at 37 C causes decreased protein synthesis Unexpected bands Denaturing temperature Denature sample at a lower tem present at higher mole is too high perature e g 60 80 C for cular weights or bands 10 15 minutes stuck in stacking gel Unexpected bands Proteolysis of translation Add protease inhibitors such as present on the gel product a2 macroglobulin leupeptin or chymostatin 0 5 1 g ml More than one peptide is Leaky scanning for translation initi translated from the ation can result in translation initi template ating at internal methionines Optimizing the Mg2 or K concen tration can increase fidelity 24 The 89S methionine T
6. 2 Load the sample from Step 1 on an SDS PAGE gel 3 Peform electrophoresis using standard conditions for your apparatus Typically electrophoresis is carried out at a constant current of 20mA Electrophoresis usually is performed until the bromophenol blue dye has run to the bottom of the gel Fluorescent Detection Materials to Be Supplied by the User e Fluorescent Imaging Instrument i e Fluorlmager SI or Fluorlmager 595 Molecular Dynamics both with a 499 argon laser the Tyohoon 8600 Molecular Dynamics with a 532nm excitation or the FMBIO II Hitachi with a 505 channel After electrophoresis is completed immediately place the gel in water and then complete fluorescent scanning Notes 1 Fixing polyacrylamide gels does not interfere with the detection of FluoroTect Green_ys labeled in vitro translation products although the signal intensity may be somewhat decreased 2 Drying fixed polyacrylamide gels in cellophane does not interfere with the detection of FluoroTect Green_tys labeled in vitro translation products although signal intensity may be somewhat decreased 3 Fixing and or drying gels may decrease the signal intensity of prestained molecular weight markers making them difficult to detect with fluorescent scanners Immunoprecipitation and Western Blot Analysis Anti BODIPY FL is available from Molecular Probes Molecular Probes Cat A 5 70 for immunoprecipitation and western blot ana
7. 2516 Internet www promega com Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 9 02 Page 25
8. FluoroTect Green_ys tRNA see Section VII C for Transcend tRNA reactions see Section VII D Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Page 8 Printed in USA Revised 9 02 C Notes 1 PCR generated templates can be used directly from the amplification reaction We recommend using 2 5 5ul from the amplification reaction but up to 7ul can be used in a 50ul reaction For PCR generated DNA that has been purified following amplification we recommend using 100 800ng of the purified product for each reaction 2 We recommend using ipl of the T7 TNT PCR Enhancer in a 50ul reaction to increase transcription translation when using PCR generated DNA 3 The TNT Quick Master Mix is designed to give the highest expression for most expression constructs However we have observed that certain gene constructs may differ in the Mg2 and K concentrations required for optimal expression in the coupled reaction For example some viral leaders will increase translation efficiency and fidelity if additional magnesium acetate and potassium chloride are added to the TNT Quick reaction If using a construct with a viral leader we suggest adding 1 2ul of the T7 TNT PCR Enhancer 4 We recommend using a grade of S9S methionine such as Amersham Biosciences Redivue L 89S methionine Amersham Biosci
9. The other components can be thawed at room temperature and then stored on ice 2 Following the example below assemble the reaction components in a 0 5ml or 1 5ml microcentrifuge tube After addition of all the components gently mix by pipetting If necessary centrifuge briefly to return the reaction to the bottom of the tube For additional information on performing a TNT Quick reaction see Notes 1 9 in Section IV C 3 Werecommend including a control reaction containing no added DNA This reac tion allows measurement of any background incorporation of labeled amino acids Example of a TNT Quick Reaction Using Plasmid DNA Standard Standard Standard Reaction Using Reaction Using Reaction Using Transcend FluoroTect Components 35S methionine tRNA Green ys tRNA TNT Quick Master Mix see Note 3 Section IV C 40ul 40ul 40ul Methionine 1mM mix gently prior to use Tul Tul SSS methionine 1 000Ci mmol at 10mCi ml see Note 4 Section IV C 2l plasmid DNA template s 0 5ug pl see Note 6 Section IV C 2Qul Qul 2Qul Transcend Biotin Lysyl tRNA see Note 9 Section IV C 1 2ul FluoroTect GreenLys tRNA 1 2u l see Note 9 Section IV C Nuclease Free Water to a final volume of 50ul 50ul 50ul Incubate the reaction at 30 C for 60 90 minutes 5 Analyze the results of translation Procedures for determination of radiolabel incorporation Section VII A and SDS PAGE analys
10. research Among other applications translation systems are used to rapidly characterize plasmid clones study structural mutations and examine translational signals Two basic approaches to in vitro protein synthesis are available 1 systems pro grammed with RNA translation systems or 2 systems programmed with DNA cou pled transcription translation systems Several general considerations to assist you in selecting the appropriate Promega product s are discussed in this section Translation Systems A number of cell free translation systems have been developed for the translation of mRNA isolated from tissue or generated in vitro Promega offers several Rabbit Reticulocyte Lysate a d e and Wheat Germ Extract Systems All are reliable convenient and easy to use systems to initiate translation and produce full size polypeptide prod ucts Rabbit Reticulocyte Lysate is appropriate for the translation of larger MRNA species and generally is recommended when microsomal membranes are to be added for cotranslational processing of translation products Promega s Flexi Rabbit Reticulocyte Lysate a 4 e is recommended where optimization of translation of particular RNAs through adjustments to salt and DTT concentrations is required Wheat Germ Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Printed in USA Page
11. 045 Revised 9 02 Page 13 Note The use of gel sys tems other than tris glycine may cause different migra tion patterns for the expressed and background bands Note The Storm instru ment Molecular Dynamics is not recommended for use with the Fluoro Tect System due to reduced sensitivity O Use gloves when handling the gels included with commercial devices and can be found in references 17 19 20 and 21 A general discussion of Western blotting with PVDF membranes is found in reference 22 PVDF membranes must be prewet in methanol or ethanol before equilibrating in transfer buffer The blot may then be sub jected to immunodetection analysis For more information refer to Promega s Protocols and Applications Guide Third Edition 23 Denaturing Gel Analysis of Translation Products Labeled with the FluoroTect Green_ys in vitro Translation Labeling System The fluorescent translation product should be resolved by running a sample on an SDS PAGE and then visualized by placing the gel on a laser based fluores cence scanning device Denaturing Gel Analysis 1 Once the translation reaction is complete or at any desired time point remove a 5ul aliquot and add it to 20ul of 1X SDS gel loading buffer Store the remainder of the translation reaction at 20 C The FluoroTect tRNA fluorophore is sensitive to extreme heating If heating to denature the pro teins do not exceed 70 C for more than 2 3 minutes
12. 1 5 650 289 and 5 814 471 Australian Pat No 649289 European Pat No 0 553 234 and Japanese Pat No 317595 have been issued to Promega Corporation for a firefly luciferase assay method which affords greater light output with improved kinetics as compared to the conventional assay Other patents are pending U S Pat Nos 5 324 637 5 492 817 and 5 665 563 European Pat No 0 566 714 B1 Australian Pat No 660329 and Japanese Pat No 2904583 have been issued to Promega Corporation for coupled transcription translation systems that use RNA polymerases and eukaryotic lysates U S Pat No 5 552 302 European Pat No 0 422 217 Australian Pat No 646803 and Japanese Pat No 3009458 have been issued to Promega Corporation for the methods and compositions for production of human recombinant placental ribonuclease inhibitor The method of recombinant expression of Coleoptera luciferase is covered by U S Pat Nos 5 583 024 5 674 713 and 5 700 673 U S Pat Nos 4 966 964 5 019 556 and 5 266 687 and Australian Pat Nos 616881 and 641261 and other pending and issued patents which claim vectors encoding a portion of human placental ribonuclease inhibitor are exclusively licensed to Promega Corporation The PCR process is covered by patents issued and applicable in certain countries Promega does not encourage or support the unauthorized or unlicensed use of the PCR process 9 Certain applications of this product may require lic
13. 22 Revised 9 02 Extract is recommended for translation of RNA preparations containing low con centrations of double stranded RNA dsRNA or oxidized thiols which are inhibitory to reticulocyte lysate Coupled Transcription Translation Systems DNA sequences cloned in plasmid vectors also may be expressed directly using either Promega s TNT Coupled Reticulocyte Lysate Systems Wheat Germ Extract Systems or E coli S30 Extract Systems a The TNT Systems are used to direct eukaryotic translation whereas the S30 Systems are under prokaryotic translational controls The TNT Systems require plasmid constructs containing a phage RNA polymerase promoter SP6 T3 or T7 for the initiation of transcription but translation in this system is under eukaryotic controls Optimal translation will occur if the AUG initiation codon is in a Kozak consen sus context A GCCAUGG 25 in the absence of inhibiting secondary struc ture The template DNA to be expressed in the S30 Systems must contain E coli promoter sequences or a phage T7 promoter sequence and prokaryotic ribo some binding sites GGAGG for translation The TNT and E coli S30 Systems can use either circular or linear DNA templates TNT Coupled Reticulocyte Lysate Systems Product Size Cat TNT SP6 Coupled Reticulocyte Lysate System a b d e 40 reactions L4600 TNT T7 Coupled Reticulocyte Lysate System a 4 e 40 reactions L4610 TNT T3 Coupled Reticulocyte Lysate
14. NA 4747bp 1917VA04_6A Figure 3 Luciferase SP6 Control DNA circle map and sequence reference points Additional descrip tion Amp B lactamase gene resistant to ampicillin ori origin of plasmid replication Sequence reference points SP6 RNA polymerase initiation 1 GL Primer 2 49 71 Luciferase gene 48 1700 Poly A dA 30 1767 1796 pUC M13 reverse primer 17mer 1833 1817 pUC M13 reverse primer 22mer 1838 1817 B lactamase gene Ampr 3838 2978 SP6 RNA polymerase promoter primer 4731 1 SP6 RNA polymerase promoter 4731 3 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 9 02 Page 21 Sac 61 Luciferase T7 Xmn Control DNA 3266 4365 bp Hind III 1560 Bol l T7 Initiation 2787 T7 Promoter 1916VA09 7A Figure 4 Luciferase T7 Control DNA circle map and sequence reference points Additional description Amp B lactamase gene resistant to ampicillin ori origin of plasmid replication Sequence reference points T7 RNA polymerase initiation 1 GI Primer 2 52 74 Luciferase gene 51 1700 Poly A dA 30 1770 1799 B lactamase gene Amp 2444 3304 T7 RNA polymerase promoter 4315 3 T7 RNA polymerase promoter Primer 4315 3 C Related Products General Considerations The in vitro synthesis of proteins is a popular method in biological
15. Protein and nucleic acid blotting and immunobiochemical detection Bio Techniques 3 276 Hicks D et al 1986 Immobilon PVDF Transfer Membrane A new membrane sub strate for Western blotting of proteins Bio Techniques 4 272 Protocols and Applications Guide Third Edition 1996 Promega Corporation Hurst R et al 1996 The TNT T7 Quick Coupled Transcription Translation System Promega Notes 58 8 Kozak M 1986 Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes Cell 44 283 92 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 9 02 Part TM045 Page 19 O Promega xI Appendix A Composition of Buffers and Solutions acrylamide solution 30 30g acrylamide 0 8g bisacrylamide Add water to a final volume of 100ml Store at 4 C fixing solution 50 methanol 10 glacial acetic acid 40 water 1X SDS gel loading buffer 50mM Tris HCI pH 6 8 100mM dithiothreitol 2 SDS 0 1 bromophenol blue 10 glycerol 1X SDS gel loading buffer lacking dithio threitol can be stored at room temperature Dithiothreitol should be added from a 1M stock just before the buffer is used SDS polyacrylamide running 10X buffer 30g Tris base 144g glycine 100ml 10 SDS Add deionized water to a fi
16. System a b d e 40 reactions L4950 TNT T7 T3 Coupled Reticulocyte Lysate System a b d e 40 reactions L5010 TNT T7 SP6 Coupled Reticulocyte Lysate System a b d e 40 reactions L5020 TNT T7 Quick for PCR DNA b c e 40 reactions L5540 TNT SP6 Coupled Reticulocyte Lysate System Trial Size a b d e 8 reactions L4601 TNT T7 Coupled Reticulocyte Lysate System Trial Size a b d e 8 reactions L4611 For Laboratory Use TNT Coupled Wheat Germ Extract Systems Product Size Cat TNTE T3 Coupled Wheat Germ Extract System a d e 40 reactions L4120 TNT SP6 Coupled Wheat Germ Extract System a b d e 40 reactions L4130 TNT T7 Coupled Wheat Germ Extract System a b d e 40 reactions L4140 TNT T7 SP6 Coupled Wheat Germ Extract System a d e 40 reactions L5030 TNT T7 T3 Coupled Wheat Germ Extract System a 5 4 e 40 reactions L5040 For Laboratory Use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 9 02 Page 23 Promega Rabbit Reticulocyte Lysate Product Size Cat Rabbit Reticulocyte Lysate Nuclease Treated a 4 e 30 reactions L4960 Rabbit Reticulocyte Lysate Untreated 1ml L4151 For Laboratory Use Bulk Rabbit Reticulocyte Lysate is available from Promega Flexi Rabbit Reticulocyte Lysate System Product Size Cat Flexi Rabbit Reticulocyte Lysate System a 4
17. T OSEE ESNet 20 A Composition of Buffers and Solutions 2 0 0 cccccseeeeeceeeeeeeeeeeeeeeseaeeeeeeeeeeaas 20 B Luciferase SP6 T7 Control DNAS cccccceccceeceeeeeeeeeseeeeseeeeceesaeaeeeeseaaeeees 21 O Related ProdUCiS creniero enan AO EE E 22 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 9 02 Page 1 Promega Q PCR Generated fragments are not recom mended for use with the SP6 promoter Use the T7 promoter Description The TNT Quick Coupled Transcription Translation Systems a b c d e are convenient single tube coupled transcription translation reactions for eukaryotic in vitro translation The original TNT Coupled Reticulocyte Lysate Systems d e simplified the process and reduced the time required to obtain in vitro translation results compared with stan dard rabbit reticulocyte lysate systems 1 Standard rabbit reticulocyte systems com monly use RNA synthesized in vitro from SP6 T3 or T7 RNA polymerase 1 The TNT Quick Coupled Transcription Translation System further simplifies the process by com bining the RNA Polymerase nucleotides salts and Recombinant RNasin Ribonuclease Inhibitor with the reticulocyte lysate solution to form a single TNT Quick Master Mix Figure 1 For most gene constructs the TNT Quick reaction pro
18. a standard Coomassie destaining solution 50 methanol 7 9 glacial acetic acid or in water for 15 30 minutes prior to drying Too much protein loaded Check the amount of samples on the gel loaded on the gel and the amount of loading buffer Too much protein loaded can cause smearing Acrylamide concentration Acrylamide concentration can be in the gel is too low increased to 12 sample contains ethanol Ethanol can cause gel smearing X References 1 Pelham H R B and Jackson R J 1976 An efficient mRNA dependent transla tion system from reticulocyte lysates Eur J Biochem 67 247 56 Bibliography of References using the TNT Coupled Transcription Translation Systems BL001 1996 Promega Corporation Chinnaiyan A M et al 1995 FADD a novel death domain containing protein interacts with the death domain of Fas and initiates apoptosis Cell 81 505 12 Cowell I and Hurst H 1996 Protein protein interaction between the transcriptional repressor E4BP4 and the TBP binding protein Dr1 Nucl Acids Res 24 3607 13 Sharp T V Witzel J E and Jagus R 1997 Homologous regions of the alpha subunit of eukaryotic translational initiation factor 2 elF2alpha and the vaccinia virus K3L gene product interact with the same domain within the dSRNA acti vated protein kinase PKR Eur J Biochem 250 85 91 Jagus R and Beckler G S 1998 Overview of eukaryotic in vitro translation and expression sy
19. cludes 1 6ml TNT Quick Master Mix 8 x 200uI e 5ug SP6 or T7 Luciferase Control DNA 0 5ug ul 0 100ul T7 TNT PCR Enhancer L1170 only e 50ul Methionine 1mM 250ul Luciferase Assay Reagent e 1 25mi Nuclease Free Water e 1 Protocol Product Cat TNT T7 Quick Coupled Transcription Translation System Trial Size L1171 TNT SP6 Quick Coupled Transcription Translation System Trial Size L2081 For Laboratory Use Each system contains sufficient reagents to perform approximately 5 x 50ul translationreac tions Includes e 200ul TNT Quick Master Mix e Sug SP6 or T7 Luciferase Control DNA 0 5yg ul 100ul T7 TNT PCR Enhancer L1171 only e 50ul Methionine 1mM e 1 Protocol Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Page 4 Printed in USA Revised 9 02 Storage and Stability Store all components at 70 C Store the Luciferase Assay Reagent in aliquots under standard reaction conditions each control reaction will require 50ul of the Luciferase Assay Reagent The Luciferase Assay Reagent is stable for 1 month when stored at 20 C or for at least 1 year at 70 C Note that the systems are shipped in foil packaging due to sensitivity of the system to carbon dioxide released from dry ice If storing the system in a freezer containing dry ice keep system compon
20. cseceeceeeeeeseeeeeeeeeeeeeeeeeeeeeeaes 10 B Non Radioactive Luciferase Control REaction ccccccccssseceeeeeseeeeseaeeeees 10 VI Cotranslational Processing Using Canine Pancreatic Microsomal Membranes ccccccseecceeeeeeeeeeseeeeseeeeseeeesaeeees 10 A General Protocol for Translation with Microsomal Membrane6 006 11 VII Post Translational Analysis ccccccccccseceeeseeeeeeee cesses eeesaeseesseneesaeeeeesaaes 12 A Determination of Percent Incorporation of Radioactive Label 0 12 B Denaturing Gel Analysis of Radioactively Labeled Translation Products 12 C Denaturing Gel Analysis of Translation Products Labeled with the Fluoro Tect Greens invitro Translation System sideniviararwnayicracomeniantoimaactea 14 D Denaturing Gel Analysis of Translation Products Labeled using the Transcend Non Radioactive Translation Detection SystemS 0000 15 Vill Positive Control Luciferase Assays cccccccccceeeeceeeeseseeeeeeeeesaaeeeeeeeeeeas 16 A Using a UNOPS Ol sete cass ceate re Sas ceed asnarin ridia aidi EEA Dasi 16 B Using a Scintillation Counter saisinncaenincsconiesdeinaaneiawnoneinianeescdnbaskeirnsapsdnenenabsainn 16 IX TroubleShoOoting ccccccccecccceeeececeseceeeeeeeeseeeeeseueeeeseeeeesaueeesseeeeesaeeeesaeeeeeas 17 X Relerent sS acerina en se eee vse gee ce tee aE NART 18 Xe AppendD cec E ar EaI SE APTEN
21. d in Promega s Protocols and Applications Guide 23 4 Perform electrophoresis using standard conditions for your apparatus Typically electrophoresis is carried out at a constant current of 20mA Electrophoresis usually is performed until the bromophenol blue dye has run off the bottom of the gel Note If a gene product is weakly expressed or contains few lysines up to 2ul of the translation reaction Reticulocyte Lysate can be loaded on an SDS gel without the loss of resolution observed with autoradiography However loading more of the translation reaction can result in high back ground on the blot Electroblotting of Proteins to Membrane For colorimetric detection see Transcend Non Radioactive Translation Detection Systems Technical Bulletin 1B182 Section V C The translation prod ucts can be blotted from the SDS polyacrylamide gel to in decreasing order of preference PVDF nitrocellulose or another membrane using any standard apparatus and protocol including semi dry systems Detailed procedures for electrophoretic blotting are usually included with commercial devices We rou tinely transfer at a constant voltage of 100V for 60 minutes using a minigel size electroblotting unit or 15 minutes using a semi dry system PVDF membrane must be pre wet in methanol before it is equilibrated in transfer buffer Instructions for chemiluminescent detection of products are found in the Transcend Non Radioactive Translation Detecti
22. duces significantly more protein 2 to 6 fold in a 60 to 90 minute reaction than a stan dard in vitro rabbit reticulocyte lysate reaction using RNA templates The TNT Quick Coupled Transcription Translation System is available in two configura tions for transcription and translation of genes cloned downstream from either the T7 or SP6 RNA polymerase promoters To use these systems 0 2 2 0ug of circular plasmid DNA containing a T7 or SP6 promoter or a PCR generated fragment containing a T7 promoter is added to an aliquot of the TNT Quick Master Mix and incubated in a 50 reaction volume for 60 90 minutes at 30 C The synthesized proteins are then analyzed by SDS polyacrylamide gel electrophoresis SDS PAGE and detected Included with the TNT Quick System is a luciferase encoding control plasmid and Luciferase Assay Reagent 9 which can be used in a non radioactive assay for rapid lt 30 seconds detection of functionally active luciferase protein Starting with either circular plasmid DNA or PCR generated DNA in vitro transcription translation results may be obtained easily in 5 6 hours TNT Coupled Reticulocyte TNT Quick Coupled Lysate System Transcription Translation System TNT Rabbit 7 Reticulocyte Lysate Add TNTE Reaction Buffer V TNT Quick Master Mix Add TNT 7 J RNA Polymerase 4 Add Amino Acid Mixture Minus Methionine Add RNasin 7 Ribonuclease Inhibitor CS Add label of choic
23. e J Add DNA template and Nuclease Free Water J Incubate at 30 C for 60 90 minutes J Separate translation products by SDS PAGE J Detect 1507MA07 Figure 1 Comparison of the TNT Coupled Reticulocyte Lysate System and the TNT Quick Coupled Transcription Translation System protocols Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Page 2 Printed in USA Revised 9 02 In addition to verifying the expected molecular weight of a gene construct the TNT Quick System is ideal for screening large numbers of constructs for either naturally occurring or deliberately engineered mutations Applications of the system include e Truncation mutation analysis e g the Protein Truncation Test PTT 2 e Drug screening affecting translation rates e Mutation and detection analysis i e enzyme kinetics e Protein protein interactions using GST pulldowns e Immunoprecipitation of protein complexes e Protein dimerization assays Ligand binding region determination confirmation competition assays In vitro expression cloning IVEC functional genomics Protein structure analysis Electrophoretic mobility shift assays EMSAs for DNA protein interactions DNA footprinting and protein cross linking studies Protein RNA binding assays Post translational modification tests V
24. e than 1ug of plasmid does not necessarily increase the amount of protein produced 7 Avoid adding calcium to the transcription translation reaction Calcium may reac tivate the micrococcal nuclease used to destroy endogenous RNA in the Master Mix and result in degradation of DNA or RNA templates 8 The TNT Quick Master Mix contains roughly 100 200mg ml of endogenous protein The level of added Transcend tRNA and FluoroTect Green_ys tRNA can be increased 1 4ul to allow more sensitive detection of proteins that contain few lysines or are poorly expressed Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 9 02 Page 9 For Assistance VI in troubleshooting Microsomal Membrane translation reactions con tact Promega s Technical Services Email techserv promega com Positive Control Translation Reactions Using Luciferase The assay for firefly luciferase activity is extremely sensitive rapid and easy to per form It is a good control for in vitro translations because only full length luciferase is active Additionally luciferase is a monomeric protein 61kDa that does not require post translational processing or modification for enzymatic activity Promega s Luciferase Assay System a 9 is a substantial improvement over conventional meth ods in both sensitivi
25. e to use the purchased product for purposes other than the internal research of the purchaser contact the Director of Licensing Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 1996 2002 Promega Corporation All Rights Reserved Flexi RNasin TNT and Wizard are trademarks of Promega Corporation and are registered with the U S Patent and Trademark Office FluoroTect and Transcend are trademarks of Promega Corporation Amplify and Redivue are trademarks of Amersham Biosciences Ltd Bio Rad is a registered trademark of Bio Rad Laboratories Inc BODIPY is a registered trademark of Molecular Probes Coomassie is a registered trademark of Imperial Chemical Industries Ltd Difco is a registered trademark of Difco Inc Fluorlmager Storm and Typhoon are registered trademarks of Molecular Dynamics FMBio is a registered trademark of Hitachi Corporation Immobilon is a trademark of Millipore Corporation NOVEX is a trademark of Novel Experimental Technology Whatman is a registered trademark of Whatman Paper Company Ltd X OMAT is a registered trademark of Eastman Kodak Co All prices and specifications are subject to change without prior notice i l l l Promega Corporation Product claims are subject to change Please contact Promega Technical Services or access the 5800 Woods Hollow Road Promega online catalog for the most up to date information on Promega products Madison WI 53711 5399 USA Telephone 608 274 4330 Fax 608 277
26. ectors e g for a 500bp PCR product use 100 800ng for each 50ul TNT Quick reaction 2 PCR products can be used directly up to 7ul or cleaned up using either the Wizard PCR Preps DNA Purification System the Wizard SV Gel and PCR Clean Up System or a standard ethanol precipitation and wash B Creating a Ribonuclease Free Environment To reduce the chance of RNase contamination gloves should be worn when set ting up experiments and microcentrifuge tubes and pipette tips should be RNase free It is not necessary to add Recombinant RNasin Ribonuclease Inhibitor to the TNT Quick reactions to prevent degradation of RNA because it is already present in the TNT Quick Master Mix C Handling of Lysate Except for the actual transcription translation incubation all handling of the TNT Quick Master Mix should be done at 4 C Any unused Master Mix should be refrozen as soon as possible after thawing to minimize loss of translational activity see Note 5 Section IV C Do not freeze thaw the Master Mix more than two times IV Translation Procedure The following is a general guideline for setting up a transcription translation reaction Also provided are examples of standard reactions using 8 S methionine radioac tive Transcend Non Radioactive Detection System colorimetric or chemilumines cent or FluoroTect Greentys Systems M fluorescent Using the Transcend Systems biotinylated lysine residues are inc
27. ences Cat AG1094 which does not cause the background labeling of the rabbit reticulo cyte lysate 42kDa protein Background labeling of the 42kDa protein can occur using other grades of label 14 In addition a stabilizer has been added to the Redivue 85S methionine to increase the stability of this product over conven tional radiolabeled amino acids so that the release of volatile gases is reduced substantially This S9S methionine may be stored at 4 C without dispensing into aliquots Other types of 3 S labeled amino acids may be oxidized easily to trans lation inhibiting sulfoxides and should be stored in aliquots at 70 C in buffer containing DTT Between 10 40uUCi 1 4ul of 85S methionine can be added to the TNT Quick reactions depending upon the balance between labeling efficiency and cost For gene constructs that express well and contain several methionines the 10uCi level 1ul is sufficient for adequate detection 5 Except for the actual transcription translation incubation all handling of the TNT Quick System components should be done at 4 C or on ice Optimum results are obtained when any unused Master Mix is quick frozen with liquid nitrogen as soon as possible after thawing to minimize loss of translational activity 6 For most plasmid constructs optimal results are obtained when 1ug of plasmid DNA template is used We recommend using 0 2 2 0ug of plasmid DNA in TNT Quick reactions The use of mor
28. enses from others h FluoroTect Green_ys incorporates dye conjugates made with the BODIPY FL fluorescent reactive dyes which are licensed from Molecular Probes Inc under U S Pat Nos 4 774 339 5 274 113 and 5 433 896 for IVE analysis for research use only including GPR and ASR applica tions and Fluorotag technology BODIPY is a registered trademark of Molecular Probes Inc and Fluorotag is a registered trademark of AmberGen Inc Licensed under U S Pat No 5 075 430 0U S Pat Nos 5 658 548 and 5 808 041 and Australian Pat No 689815 have been issued to Promega Corporation for nucleic acid purification on silica gel and glass mixtures Other patents pending K U S Pat No 5 981 235 and Australian Pat No 729932 have been issued to Promega Corporation for methods for isolating nucleic acids using alkaline protease Other patents are pending Australian Pat No 730718 and Singapore Pat No 64532 has been issued to Promega Corporation for an improved filtration system and method Other patents are pending m NOTICE TO PURCHASER LIMITED USE LICENSE This product is sold under licensing arrangements between Promega Corporation and Invitrogen Corporation The purchase price of this prod uct includes limited nontransferable rights under U S Pat Nos 5 082 784 and 5 192 675 owned by Invitrogen Corporation to use the product only for the internal research purposes of the purchaser For information on purchasing a licens
29. ents sealed in foil packaging for best results DO NOT store the unfoiled lysate in the presence of dry ice Prolonged exposure to dry ice causes significant loss of activity The expiration date for the TNT Quick Master Mix is listed on the product vial Do not freeze thaw the Master Mix more than 2 times Avoid multiple freeze thaw cycles or exposure to frequent temperature changes as these fluctuations can greatly alter product stability lll General Considerations A DNA Template Considerations DNA Expression Elements 1 In addition to circular plasmid DNA PCR generated DNA templates can be transcribed translated using the T7 System For maximal expression from such templates it is recommended that approximately 11bp be present upstream of the T7 RNA polymerase promoter for efficient promoter binding A stop codon usually UAA is important for truncated gene products in order to prevent ribosomes from stalling at the ends of RNAs without stop codons This can be done through appropriate primer design The best transcription translation results are obtained when the fragment contains the T7 RNA polymerase promoter We do not recommend using linear DNA with the SP6 System because of reduced tanscription efficiencies 2 While Rabbit Reticulocyte Lysate based systems are less sensitive to 5 untranslated region UTR secondary structure than other systems it is still important to avoid strong hairpin secondary structure in the 5 UTR regio
30. erification characterization of cloned genes The TNT Quick Coupled Transcription Translation Systems are also useful for detecting protein protein interactions in vitro Proteins labeled using TNT Quick Coupled Transcription Translation System can be used as probes to detect interactions with sus pected protein partners that have been expressed as GST glutathione S transferase or epitope tagged fusion proteins 3 S gt S methionine labeled proteins can be synthesized using coupled in vitro reactions from either full length cDNAs or deletion mutants The fusion proteins can be bound to an affinity matrix along with the radioactive proteins with which they interact 4 6 The bound radioactive proteins are then eluted and analyzed by SDS PAGE or Western analysis Figure 2 6 The fusion tag approach has been used to study receptor mediated control of apoptosis 7 Alternatively a non radioactive approach may be used the protein is labeled with biotiny lated lysine e g Transcend Biotinylated tRNA or is fluorescently tagged e g FluoroTect Green_ys BODIPY FL labeled tRNA and combined with a GST tagged protein The biotinylated protein is detected by methods similar to those used in Western blotting 8 9 For a complete list of references for these and other applications see reference 6 or visit Promega s Technical Resource Center citations at www promega com citations Promega Corporation 2800 Woods Hollow Road
31. f R C 1985 A novel transcription property of SP6 and T7 RNA polymerases Dependence on template structure Nucl Acids Res 13 6223 36 Jackson R J and Hunt T 1983 Preparation and use of nuclease treated rabbit reticulo cyte lysates for the translation of eukaryotic messenger RNA Meth Enzymol 96 50 74 Wood K V 1991 Recent advances and prospects for use of beetle luciferases as genetic reporters In Bioluminescence and Chemiluminescence Current Status Stanley P E and Kricka J eds John Wiley and Sons Chichester N Y Andrews D 1987 Assaying protein translocation across the endoplasmic reticulum membrane Promega Notes 11 1 Towbin H et al 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitro cellulose sheets Procedure and some applications Proc Natl Acad Sci USA 76 4350 4 Burnette W N 1981 Western blotting Electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detec tion with antibody and radioiodinated protein A Anal Biochem 112 195 203 Bittner M et al 1980 Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyI cellulose or nitrocellulose sheets Anal Biochem 102 459 71 Towbin H and Gordon J 1984 Immunoblotting and dot immunobinding current sta tus and outlook J Immunol Meth 72 313 20 Bers G and Garfin D 1985
32. for 10 minutes At the end of the incubation add 900ul of ice cold 25 TCA 2 casamino acids to precipitate the translation product Incubate on ice for 30 minutes Wet a Whatman GF A glass fiber filter with a small amount of ice cold 5 TCA Collect the precipitated translation product by vacuum filtering 250ul of the TCA reaction mix Rinse the filter 3 times with 1 3ml of ice cold 5 TCA Rinse once with 1 3ml of acetone Allow the filter to dry at room temperature or under a heat lamp for at least 10 minutes For determination of 39S incorporation put the filter in the appropriate scin tillation cocktail invert to mix and count in a liquid scintillation counter To determine total counts present in the reaction spot a 5ul aliquot of the TCA reaction mix directly onto a filter Dry the filter for 10 minutes Count in a liquid scintillation counter as in Step 5 To determine background counts remove 2ul from a 50ul translation reac tion containing no DNA and proceed as described in Steps 1 5 Perform the following calculation to determine percent incorporation cpm of washed filter Step 5 100 bi alice com of unwashed filter Step 6 x 50 percent incorporati Perform the following calculation to determine the fold stimulation over background com of washed filter Step 5 fold stimulation cpm of no DNA control reaction filter Step 7 B Denaturing Gel Analysis of Radioactively Labeled Translation Products
33. g Transcend FluoroTect Components 35S methionine tRNA GreenLys tRNA TNT T7 Quick Master Mix see Note 3 Section IV C 40ul 40ul 40ul Methionine 1mM mix gently prior to use Tul Tul S5S methionine 1 000Ci mmol at 10mCi ml see Note 4 Section IV C 2l PCR generated DNA template s see Note 1 Section IV C 2 5 5ul 2 5 5ul 2 5 5ul T7 TNT PCR Enhancer see Note 2 Section IV C Tul Tul Tul Transcend Biotin Lysyl tRNA see Note 9 Section IV C 1 2ul Fluoro Tect Greengys tRNA 1 2ul see Note 9 Section IV C Nuclease Free Water to a final volume of 50ul 50ul 50ul 4 Incubate the reaction at 30 C for 60 90 minutes 5 Analyze the results of translation Procedures for determination of radiolabel Nuclease Free Water Cat P1193 radiolabeled amino acid for radioactive detection Note 4 Seciton IV C or Transcend tRNA Cat L5061 or Transcend Colorimetric Cat L5070 or Chemiluminescent Cat L5080 Translation Detection System for non radioactive detection or Fluoro Tect Green_ys in vitro Translation Labeling System for fluorescent detection Cat L5001 Remove the reagents from storage at 70 C Rapidly thaw the TNT Quick Master Mix by hand warming and place on ice The other components can be thawed at room temperature and then stored on ice incorporation Section VII A and SDS PAGE analysis of translation products Section VII B are provided If using
34. here are reports of a 42kDa band used is not of translation with some grades of S S methio al grade or beyond its nine 14 We recommend expiration date Amersham Biosciences Redivue L 85S methionine Amersham Biosciences Cat AG1094 to avoid this 42kDa band Globin may appear on Globin may show on a stained gel the autoradiogram or and occasionally on the auto stained gel radiogram It appears as a broad band migrating at 10 15kDa For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail techserv promega com Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 9 02 Part TM045 Page 17 Troubleshooting continued Symptoms Possible Causes Comments Unexpected bands Aminoacyl tRNAs may Add RNase A to the lysate reac present on the gel produce background tion after completion to a final continued bands 25kDa concentration of 0 2mg ml Incubate for 5 minutes at 30 C Oxidized B mercapto Use a loading buffer that contains ethanol is present or not 2 SDS and 100mM DTT enough SDS in the loading buffer There is smearing on Gel not clean Gel must be washed before plac the gel ing onto film Once gel electro phoresis is complete soak the gel in either
35. is of translation products Section VII B are provided O Note Technical Manuals and Bulletins are available online at www promega com tbs or by request from Technical Services Note Small scale reac tions may be performed by reducing the recom mended volumes propor tionally Note The optimal cou pled transcription transla tion reaction occurs in 60 90 minutes at 30 C Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 9 02 Part TM045 Page 7 Note Small scale reac tions may be performed by reducing the recom mended volumes propor tionally B General Protocol for TNT T7 Quick Coupled Transcription Translation Reactions Using PCR Generated DNA Materials to Be Supplied by the User 2 Following the example below assemble the reaction components in a 0 5ml or 1 5ml microcentrifuge tube After addition of all the components gently mix by pipetting If necessary centrifuge briefly to return the reaction to the bottom of the tube For additional information on performing a TNT Quick reaction see Notes 1 9 in Section IV C 3 Werecommend including a control reaction containing no added DNA This reac tion allows measurement of any background incorporation of labeled amino acids Standard Standard Standard Reaction Using Reaction Using Reaction Usin
36. its of sensitivity may vary depending upon the particular instrument used The assay should be linear in some portion of the detection range of the instrument Please consult your instru ment operator s manual for general operating instructions A Using a Luminometer 1 Dispense 50ul of the Luciferase Assay Reagent into luminometer tubes one tube per sample 2 Program the luminometer to perform a 2 second measurement delay followed by a 10 second measurement read for luciferase activity The read time may be shortened if sufficient light is produced 3 Add 2 5ul of cell lysate to a luminometer tube containing the Luciferase Assay Reagent Mix by pipetting 2 3 times or vortex briefly 4 Place the tube in the luminometer and initiate reading 5 If the luminometer is not connected to a printer or computer record the reading B Using a Scintillation Counter Ideally the coincidence circuit of the scintillation counter should be turned off Usually this is achieved through an option of the programming menu or by a switch within the instrument Consult the user s manual or the manufacturer of the scintillation counter If the circuit cannot be turned off a linear relationship between luciferase concentration and cpm still can be produced by calculating the square root of measured counts per minute com minus background cpm i e sample background 2 To measure background cpm use water or Luciferase Assay Reagent as a b
37. ittle loss of luciferase activity Cotranslational Processing Using Canine Pancreatic Microsomal Membranes Microsomal vesicles are used to study cotranslational and initial post translational processing of proteins Processing events such as signal peptide cleavage membrane insertion translocation and core glycosylation can be examined by the translation of the appropriate gene in vitro in the presence of these membranes To ensure consis tent performance with minimal background Promega s Canine Pancreatic Microsomal Membranes amp Cat Y4041 have been isolated so that they are free from MRNA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Page 10 Printed in USA Revised 9 02 Materials to Be Supplied by the User e Canine Pancreatic Microsomal Membranes Cat Y4041 85S methionine 1 000Ci mmol at 10mCi ml 1 General Protocol for Translation with Microsomal Membranes Remove the reagents from the freezer and allow them to thaw on ice Promega 2 Mix the following components on ice in the order given in a sterile 1 5ml microcentrifuge tube an Note The storage buffer sSjmethionine 1 O00Cimmol at 10mCi m a E e Microsomal Membranes is see Note 4 Section IV C 2 0ul 50mM triethanolamine plasmid DNA 0 5yug 0 5ul 2mM DTT and 250mM Canine Pancreatic Microsomal Memb
38. lank Use the same protocol as luciferase assays using a luminometer Section VII A The sample may be placed directly in the scintillation vial if it completely covers the bottom of the vial clear or translucent vials are acceptable Do not add scin tillant because it will inactivate luciferase Alternatively place the sample ina microcentrifuge tube and then place the tube in the scintillation vial To ensure consistency when working with multiple samples place each microcentrifuge tube at the same relative position within the scintillation vial For consistency in measuring luciferase activity use the scintillation counter in manual mode Initiate each sample reaction immediately before measurement and read the samples one at a time Because the enzymatic reaction produces light at all wavelengths read the samples with all channels open open window To reduce background counts it may be necessary to wait 10 30 seconds before counting Read individual samples for 1 5 minutes Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Page 16 Printed in USA Revised 9 02 IX Troubleshooting Symptoms Possible Causes Comments The control reaction Loss of activity of The lysate should not be used produces no luciferase reaction components after more than two freeze thaw cycles Do not use reagents after
39. lysis of translation products Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Page 14 Printed in USA Revised 9 02 D Denaturing Gel Analysis of Translation Products Labeled Using the Transcend Non Radioactive Translation Detection Systems Biotinylated protein standards Bio Rad Cat 161 0319 can be used to determine the apparent molecular weight of the translated biotinylated protein Alternatively fluorescently labeled size standards can be observed after transfer and marked with a pencil under UV irradiation The positions of unlabeled size standards also can be determined by staining the blot after transfer See Transcend Non Radioactive Translation Detection Systems Technical Bulletin TB182 1 Once the 50ul translation reaction is complete or at any desired time point remove a 1ul aliquot and add it to 15ul of SDS sample buffer The remainder of the reaction may be stored at 20 C 2 Close the tube and heat at 90 100 C for 2 minutes to denature the proteins Note In some cases high molecular weight complexes are formed at 100 C and denaturation may need to be performed at lower temperatures e g 20 minutes at 60 C or 3 4 minutes at 80 85 C 3 Load the denatured sample on an SDS polyacrylamide gel Protocols for SDS polyacrylamide gel electrophoresis may be foun
40. mmercial reagents such as Amplify Reagent Amersham Biosciences can be used for fluorographic enhancement of signal Alternatively the fixed gel can be exposed to a phosphorimaging screen These systems provide greater sensitivity greater soeed and the ability to quantitate the radioactive bands 6 Dry the gel before exposure to film as follows Soak the gel in 7 acetic acid 7 methanol 10 glycerol for 5 minutes to prevent the gel from cracking during drying Place the gel on a sheet of Whatman 3MM filter paper cover with plastic wrap and dry at 80 C for 30 90 minutes under a vacuum using a conventional gel dryer dry completely The gel also may be dried overnight using Promega s Gel Drying Kit Cat V7120 To decrease the likelihood of cracking gradient gels dry them with the wells pointing down Expose the gel on Kodak X OMAT AR film for 1 6 hours at 70 C with fluorography or 6 15 hours at room temperature with autoradiography 7 For Western blot analysis of proteins transfer immobilize the protein from the gel onto nitrocellulose or PVDF membrane 17 18 Usually Western blots are made by electrophoretic transfer of proteins from SDS polyacry lamide gels Detailed procedures for electrophoretic blotting usually are Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM
41. n because this can impair translation efficiency 10 3 We have observed enhanced translation of proteins when using DNA con structs containing a poly A sequence downstream of the gene of interest Poly A sequences are important for MRNA stability and can play a role in translation initiation in Rabbit Reticulocyte Lysate 11 For example we have observed a 2 to 5 fold increase in the production of luciferase when the gene is cloned into the pSP64 Poly A Vector Cat P1241 Plasmid DNA 1 Residual ethanol should be removed from DNA preparations before they are added to the TNT Quick Master Mix 2 Linearized templates produced by restriction enzyme digestion should be cleaned up either by using the Wizard PCR Preps DNA Purification System or by phenol chloroform extraction followed by ethanol precipitation before use in the TNT Quick reaction 3 Plasmid DNA can be purified using the Wizard Plus i or Wizard Plus SV k Minipreps DNA Purification System DNA prepared by the standard alkaline lysis method described by Sambrook Fritsch and Maniatis 12 is also suffi ciently clean for use in the TNT Quick Coupled Transcription Translation S Do Not store the unfoiled lysate in the presence of dry ice Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 9 02 Pa
42. nal volume of 1 liter Store at room temperature separating gel 4X buffer 18 17g Tris base 4ml 10 SDS Bring the volume to approximately 80ml with deionized water Adjust to pH 8 8 with 12N HCl and add deionized water to a final vol ume of 100ml Store at room temperature stacking gel 4X buffer 6 06g Tris base 4ml 10 SDS Bring the volume to approximately 80ml with deionized water Adjust to pH 6 8 with 12N HCl and add deionized water to a final volume of 100ml Store at room temperature Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Page 20 Printed in USA Revised 9 02 B Luciferase SP6 T7 Control DNAs The Luciferase SP6 T7 Control DNAs are used as functional controls in the TNT Quick Coupled Transcription Translation System The Control DNAs contain the gene for luciferase under transcriptional control of a phage RNA polymerase promoter The constructs carry a 30bp poly d A d T tail following the luciferase gene The maps of the Luciferase SP6 Control DNA and T7 Control DNA are shown in Figures 3 and 4 respectively Please note that these vectors are intended for use as control luciferase expression vectors only They are not intended for use as cloning vectors SP6 Promoter l SP6 Initiation 1 Hind III 8 Not 21 BamH 41 eae Luciferase SP6 Control D
43. nine Microsomal Membranes in Pancreatic Microsomal Membranes will be less than the amount produced me Teachon in TNT Quick reactions alone Depending on the construct used protein syn thesis efficiency can be expected to drop between 10 50 in the presence of Microsomal Membranes 4 In some cases it is difficult to determine if efficient processing or glycosyla tion has occurred by gel analysis alone Other assays such as various pro tection assays 16 may be required to determine if processing events have taken place Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 9 02 Page 11 Vil Post Translational Analysis Materials to Be Supplied by the User Solution compositions are provided in Section XI A e 1M NaOH e 30 acrylamide solution e 25 TCA 2 casamino acids e separating gel 4X buffer Difco brand Vitamin Assay Grade e stacking gel 4X buffer e 5 TCA e SDS sample buffer e Whatman GF A glass fiber filter e SDS polyacrylamide gels Whatman Cat 1820 021 e optional precast polyacrylamide e acetone gels e Whatman 3MM filter paper A Determination of Percent Incorporation of Radioactive Label 1 After the 50ul translation reaction is complete remove 2ul from the reaction and add it to 98ul of 1M NaOH 2 H202 Vortex briefly and incubate at 37 C
44. on Systems Technical Bulletin 1TB182 Section V D Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 9 02 Page 15 O Promega Q The Luciferase Assay Reagent and samples should be at ambient temperature prior to performing a luciferase assay Do not add scintillant because it will inactivate the luciferase and is not needed VIII Positive Control Luciferase Assays Light intensity is a measure of the rate of catalysis by luciferase and is therefore dependent upon temperature The optimum temperature for luciferase activity is approximately room temperature 20 25 C It is important that the Luciferase Assay Reagent be fully equilibrated to room temperature before beginning meas urements To ensure temperature equilibration place a thawed aliquot of the Luciferase Assay Reagent in a sealed tube into a water bath maintained at ambient temperature and equilibrate for at least 30 minutes The sample to be assayed should also be at ambient temperature Either a luminometer or a scintillation counter can be used for quantitation There is usually insufficient light output for qualitative visual detection A luminometer can measure as little as 10 20 moles 0 001pg of luciferase whereas a scintillation counter typically has a less sensitive detection limit However the lim
45. onto an SDS polyacrylamide gel or stored at 20 C It is not necessary to sepa rate labeled polypeptides from free amino acids by acetone precipitation 4 Typically electrophoresis is carried out at a constant current of 15mA in the stacking gel and 30mA in the separating gel or 30mA for a gradient gel Electrophoresis is usually performed until the bromophenol blue dye has run off the bottom of the gel Disposal of unincorporated label may be easier if the gel is stopped while the dye front remains in the gel as the dye front also contains the unincorporated labeled amino acids If transferring the gel to a membrane filter for Western blotting proceed to Step 7 5 Place the polyacrylamide gel in a plastic box and cover the gel with fixing solution as prepared in Section XI A for 30 minutes Agitate slowly on an orbital shaker Pour off the fixing solution Proceed to Step 6 gel drying prior to film exposure Optional Labeled protein bands in gels may be visualized by autoradiogra phy or fluorography Fluorography dramatically increases the sensitivity of detection of 39S 14C and 3H labeled proteins and is recommended for the analysis of in vitro translation products The increased detection sensitivity of fluorography is obtained by infusing an organic scintillant into the gel The scintillant converts the emitted energy of the isotope to visible light and increases the proportion of energy that may be detected by X ray film Co
46. orporated into nascent proteins during translation This biotinylated lysine is added to the transcription translation reaction as a precharged labeled biotinylated lysine tRNA complex Transcend tRNA rather than a free amino acid For more information on the Transcend Systems request Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Printed in USA Page 6 Revised 9 02 Technical Bulletin 1B182 The FluoroTect System uses a charged lysine tRNA labeled with the fluorophore BODIPY FL to incorporate fluorescently labeled lysine residues into the in vitro translation product For more information on the FluoroTect System request Technical Bulletin TB285 A General Protocol for TNT Quick Coupled Transcription Translation Reactions Using Plasmid DNA Materials to Be Supplied by the User e Nuclease Free Water Cat P1193 e radiolabeled amino acid for radioactive detection Note 4 Seciton IV C or Transcend tRNA Cat L5061 or Transcend Colorimetric Cat L5070 or Chemiluminescent Cat L5080 Translation Detection System for non radioactive detection or Fluorolect Green_ys in vitro Translation Labeling System for fluorescent detection Cat L5001 1 Remove the reagents from storage at 70 C Rapidly thaw the TNT Quick Master Mix by hand warming and place on ice
47. ranes SUCrOSe see Note 1 below 0 3 1 8ul Nuclease Free Water to a final volume of 25ul Incubate at 30 C for 60 90 minutes Note TNT Quick 4 Analyze the results of translation and processing Procedures for incorpora Coupled Transcription tion assays Section VII A and SDS PAGE analysis of translation products Translation Systems are Section VII B are provided not tested for performance with Canine Microsomal Notes Membranes 1 The amount of Canine Microsomal Membranes used in the reaction may need to be titrated While these reaction conditions will be suitable for most applications the efficiency of processing using membranes may vary Thus Note We do NOT recom reaction parameters may need to be altered to suit individual requirements mend using Canine In general increasing the amount of membranes in the reaction increases v erosoma A the proportion of polypeptides processed but reduces the total amount of a Caine j polypeptides synthesized Transcription 2 For reactions using the TNT Quick CoupledTranscription Translation Translation Systems System the Canine Microsomal Membranes will inhibit transcription We do because SP6 polymerase not recommend exceeding 1 8ul of Canine Microsomal Membranes 5 Bera Transcription Translation may be inhibited by as much as 50 with 0 6ul of ola a ents as 70 Canine Microsomal Membranes by the presence of Canine 3 The amount of protein produced in TNT Quick reactions using Ca
48. rt TM045 Page 5 System For most constructs optimal results are obtained when 1g of plas mid DNA template is used However we have used 0 2 2 0ug of DNA tem plate and obtained satisfactory levels of translation The use of more than 1ug of plasmid does not necessarily increase the amount of protein produced 4 lf linearizing plasmid DNA for use with the T7 System avoid the use of restric tion enzymes that yield 3 overhangs PstI Kon Im Sac l Sac ll BstX Nsi Apa and Aat Il as aberrant transcription products can be produced 13 If no alternative enzyme is available the 3 overhang can be removed by the addition of Klenow DNA polymerase at a concentration of 5 units ug DNA fol lowed by incubation for 15 minutes at 22 C 5 Check the sequence of the DNA template for the presence of additional upstream start codons During translation the ribosome is thought to scan from the 5 end of the RNA and begin translation at the first AUG encoun tered Thus any AUGs within the transcribed portion of the vector or untranslated sequence of the insert may cause translation initiation to occur prior to the desired start codon and result in a shift in the reading frame or production of a larger protein than expected PCR Generated DNA Templates 1 Because PCR DNA templates are usually much smaller than plasmid tem plates the amount of DNA necessary for optimal expression is often less than for inserts cloned into plasmid v
49. stems Current Protocols in Cell Biology Bonifacirro et al eds John Wiley amp Sons Inc 11 1 1 11 1 13 Cleveland D L and Ihle J H 1995 Contenders in FasL TNF death signaling Cell 81 479 82 Pei L 1999 Pituitary tumor transforming gene protein associates with ribosomal protein S10 and a novel human homologue of DnaJ in testicular cells J Biol Chem 274 3151 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Page 18 Printed in USA Revised 9 02 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 29 Promega Chen W and Pei L 2000 A novel binding factor facilitates nuclear translocation and transcriptional activation function of the pituitary tumor transforming gene product J Biol Chem 275 19422 Frances V Morle F and Godet J 1992 Identification of two critical base pairings in 5 untranslated regions affecting translation efficiency of synthetic uncapped globin mRNAs Biochim Biophys Acta 1130 29 37 Jackson R J and Standart N 1990 Do the poly A tail and 3 untranslated region con trol mRNA translation Cell 62 15 24 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY Schenborn E T and Mierendor
50. ty and simplicity 15 The control reaction can be performed with or without the addition of radiolabeled amino acids A Radioactive Luciferase Control Reaction 1 The following example contains 8 S methionine TNT Quick Master Mix see Note 3 Section IV C 40ul 85S methionine 1 000Ci mmol at 10mCi ml see Note 4 Section IV C 2l Appropriate Luciferase Control DNA 0 5yg ul see Section XI B 2ul Nuclease Free Water to a final volume of 50ul 2 Incubate the reaction at 30 C for 60 90 minutes see Note 3 Section IV C Analyze the results of translation by measuring direct incorporation of radio label Section VII A and or gel analysis of translation products Section VII B 4 The Luciferase Control reactions can be stored at 20 C for up to 2 months or at 70 C for up to 6 months with little loss of luciferase activity B Non Radioactive Luciferase Control Reaction 1 The following example contains Methionine TNT Quick Master Mix see Note 3 Section IV C 40ul Methionine 1mM Tul Appropriate Luciferase Control DNA 0 5yg ul see Section XI B 2ul Nuclease Free Water to a final volume of 30ul 2 Incubate the translation reaction at 30 C for 60 90 minutes 3 Test for the synthesis of functional luciferase using the standard luciferase assay see Section VIII A 4 The Luciferase Control reactions can be stored at 20 C for up to 2 months or at 70 C for up to 6 months with l
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