TNT® Quick Coupled Transcription/Translation
Contents
1. 17 7 C Denaturing Gel Analysis of Radioactively Labeled Translation Products 17 7 D Denaturing Gel Analysis of Translation Products Labeled with the FluoroTect GreenLys in vitro Translation Labeling System 19 7 E Denaturing Gel Analysis of Translation Products Labeled with the Transcend Non Radioactive Translation Detection Systems 20 8 Positive Control Luciferase Assays 21 8 A Using a Luminometer 21 8 B Using a Scintillation Counter 21 9 Troubleshooting 22 10 References 24 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2512. 8 4 A General Protocol for TNT Quick Coupled Transcription Translation Reactions Using Plasmid DNA 9 4 B General Protocol for TNT T7 Quick Coupled Transcription Translation Reactions Using PCR Generated DNA 10 4 C Notes 12 5 Positive Control Translation Reactions Using Luciferase 13 5 A Radioactive Luciferase Control Reaction 13 5 B Non Radioactive Luciferase Control Reaction 13 6 Cotranslational Processing Using Canine Pancreatic Microsomal Membranes 14 6 A General Protocol for Translation with Microsomal Membranes 14 7 Post Translational Analysis 15 7 A Western Blot Analysis 16 7 B Determination of Percent Incorporation of Radioactive Label
3. 1 Fixing polyacrylamide gels does not interfere with the detection of FluoroTect GreenLys labeled in vitro translation products although the signal intensity may be somewhat decreased 2 Drying xed polyacrylamide gels in cellophane does not interfere with the detection of FluoroTect GreenLys 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 di cult to detect with uorescent scanners 20 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 7 E Denaturing Gel Analysis of Translation Products Labeled with 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 uorescently 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 50 l translation reaction is complete or at any desired time point remove a 1 l aliquot an
4. 3 We recommend including a control reaction containing no added DNA This reaction allows measurement of any background incorporation of labeled amino acids Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 11 www promega com TM045 Revised 12 13 Labeled Components Unlabeled 35S methionine Transcend tRNA FluoroTect GreenLys tRNA TNT T7 Quick Master Mix see Note 3 Section 4 C 40 l 40 l 40 l 40 l Methionine 1mM mix gently prior to use 1 l 1 l 1 l 35S methionine 1 000Ci mmol at 10mCi ml see Note 4 Section 4 C 2 l PCR generated DNA template s see Note 1 Section 4 C 2 5 5 l 2 5 5 l 2 5 5 l 2 5 5 l T7 TNT PCR Enhancer see Note 2 Section 4 C 1 l 1 l 1 l 1 l Transcend Biotin Lysyl tRNA see Note 9 Section 4 C 1 2 l FluoroTect GreenLys tRNA see Note 9 Section 4 C 1 2 l Nuclease Free Water to a nal volume of 50 l 50 l 50 l 50 l Note Small scale reactions may be performed by reducing the recommended volumes proportionally 4 Incubate the reaction at 30 C for 60 90 minutes 5 Analyze the results of translation A protocol for Western Blot analysis is provided in Section 7 A Procedures for determining radiolabel incorporation Section 7 B and SDS PAGE analysis of trans
5. 0 2 2 0 g 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 l 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 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 PCR generated fragments are not recommended for use with the SP6 promoter Use the T7 promoter 11 Appendix 26 11 A Composition of Bu ers and Solutions 26 11 B Luciferase SP6 T7 Control DNAs 27 11 C Related Products 29 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll F
6. y 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 4 C 3 We recommend including a control reaction containing no added DNA This reaction allows measurement of any background incorporation of labeled amino acids Examples of TNT Quick Reactions Using Plasmid DNA Labeled Components Unlabeled 35S methionine Transcend tRNA FluoroTect GreenLys tRNA TNT Quick Master Mix see Note 3 Section 4 C 40 l 40 l 40 l 40 l Methionine 1mM mix gently prior to use 1 l 1 l 1 l 35S methionine 1 000Ci mmol at 10mCi ml see Note 4 Section 4 C 2 l plasmid DNA template s 0 5 g l see Note 6 Section 4 C 2 l 2 l 2 l 2 l Transcend Biotin Lysyl tRNA see Note 9 Section 4 C 1 2 l FluoroTect GreenLys tRNA see Note 9 Section 4 C 1 2 l Nuclease Free Water to a nal volume of 50 l 50 l 50 l 50 l Note Small scale reactions may be performed by reducing the recommended volumes proportionally 10 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 4 A General Protocol for TNT Quick Coupled Transcription Translation Reactions Using Plasmid DNA continued 4 Incubate the reaction at 30
7. 641 and 5 650 289 b 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 A license from Promega for research reagent products and from The Regents of the University of California for all other elds is needed for any commercial sale of nucleic acid contained within or derived from this product c Any use of the product for diagnos cs requiring clearance or approval by the U S Food and Drug Administra on or a foreign equivalent may require a license under Mayo Clinic United States Patent No 6 027 913 2011 2013 Promega Corpora on All Rights Reserved Flexi RNasin TnT and Wizard are registered trademarks of Promega Corpora on FluoroTect HisLink MagneGST MagZ PureYield and Transcend are trademarks of Promega Corpora on Amplify FluorImager Storm and Typhoon are registered trademarks of GE Healthcare Bio sciences BODIPY is a registered trademark of Molecular Probes Inc Coomassie is a registered trademark of Imperial Chemical Industries Ltd EasyTag is a trademark of PerkinElmer FMBIO is a registered trademark of Hitachi So ware Engineering Company Ltd Immobilon is a trademark of Millipore Corpora on iBlot and Novex are registered trademarks of Invitrogen Corpora on Ready Gel is a registered trademark of Bio Rad Laboratories Inc X OMAT is a registered trademark of Eastman Kodak Co Products may be covered by pending or issued patents or may have
8. Immunoprecipitation of protein complexes Protein dimerization assays Ligand binding region determination con rmation 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 modi cation tests Veri cation characterization of cloned genes The TNT Quick Coupled Transcription Translation Systems are also useful for detecting protein protein interactions in vitro 35S methionine labeled proteins labeled using TNT Quick Coupled Transcription Translation System can be used as probes to detect interactions with suspected protein partners that have been expressed as GST glutathione S transferase or epitope tagged fusion proteins 3 35S 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 a nity 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 bio
9. also may be dried overnight using the 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 lm for 1 6 hours at 70 C with uorography 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 19 20 Usually Western blots are made by electrophoretic transfer of proteins from SDS polyacrylamide gels Detailed procedures for electrophoretic blotting usually are included with commercial devices and can be found in references 19 21 22 and 23 A general discussion of Western blotting with PVDF membranes is found in reference 24 PVDF membranes must be prewet in methanol or ethanol before equilibrating in transfer bu er The blot may then be subjected to immunodetection analysis For more information refer to the Promega Protocols and Applications Guide Online Edition 25 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 19 www promega com TM045 Revised 12 13 7 D Denaturing Gel Analysis of Translation Products Labeled with the FluoroTect GreenLys in vitro Translation Labeling System The uorescent translation product should be resolved by SDS PAGE and then visualized by placing the gel on a laser b
10. brie y to collect the contents in the bottom of the tube 3 Load 20 l onto a 4 20 gradient Tris glycine SDS polyacrylamide gel 4 Following electrophoresis remove the gel and place it in water 5 Transfer the proteins to a PVDF membrane using a Western blotting system e g iBlot System Invitrogen Cat IB1001 6 Block the membrane using 15ml of 5 Blot Quali ed BSA in TBST 1X TBS 0 1 Tween 20 Incubate for 1 hour with gentle shaking 7 Dilute your primary antibody in 1X TBST Note We recommend that you titrate your primary antibody dilutions to determine what dilution produces the best results for your protein 8 Following incubation remove the blocking solution from the membrane and add 15ml of diluted primary antibody 9 Incubate the membrane with the primary antibody at room temperature for 1 hour with gentle shaking 10 Remove the primary antibody solution and wash the membrane with 15ml of 1X TBST for 5 minutes with gentle shaking 11 Repeat the wash 5 more times for a total of six washes 12 Dilute your secondary antibody 1 2 500 in 1X TBST 13 Following that last wash remove bu er from the membrane and add 15ml of diluted secondary antibody 14 Incubate the membrane with the secondary antibody for 1 hour with gentle shaking 15 Following the incubation remove the secondary antibody solution and wash the membrane with 15ml of 1X TBST for ve minutes Repeat fo
11. certain limita ons Please visit our Web site for more informa on All prices and speci ca ons are subject to change without prior no ce Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date informa on on Promega products
12. presence of these membranes To ensure consistent performance with minimal background Canine Pancreatic Microsomal Membranes Cat Y4041 have been isolated so that they are free from mRNA For assistance in troubleshooting Microsomal Membrane translation reactions contact Promega Technical Services E mail techserv promega com 6 A General Protocol for Translation with Microsomal Membranes Materials to Be Supplied by the User Canine Pancreatic Microsomal Membranes Cat Y4041 35S methionine 1 000Ci mmol at 10mCi ml 1 Remove the reagents from the freezer and allow them to thaw on ice Note The storage bu er for Canine Pancreatic Microsomal Membranes is 50mM triethanolamine 2mM DTT and 250mM sucrose 2 Mix the following components on ice in the order given in a sterile 1 5ml microcentrifuge tube T7 TNT Quick Master Mix 20 l 35S methionine 1 000Ci mmol at 10mCi ml see Note 4 Section 4 C 2 0 l plasmid DNA 0 5 g l 0 5 l Canine Pancreatic Microsomal Membranes see Note 1 below 0 3 1 8 l Nuclease Free Water to a nal volume of 25 l 3 Incubate at 30 C for 60 90 minutes 4 Analyze the results of translation and processing Procedures for Western Blot analysis Section 7 A incorporation assays Section 7 B and SDS PAGE analysis of translation products Section 7 C are provided Note TNT Quick Coupled Transcription Translation Systems are not tested for performanc
13. reaction is complete or at any desired timepoint remove a 1 5 l aliquot and add it to 20 l of SDS sample bu er The remainder of the reaction may be stored at 20 C or at 70 C for long term storage 18 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 7 C Denaturing Gel Analysis of Radioactively Labeled Translation Products continued 2 Cap the tube and heat at 100 C for 2 minutes to denature the proteins This may cause protein aggregation Incubation at a lower temperature e g 20 minutes at 60 C 10 minutes at 70 C or 3 4 minutes at 80 85 C may be more appropriate 3 A small aliquot 5 10 l of the denatured sample can then be loaded onto an SDS polyacrylamide gel or stored at 20 C It is not necessary to separate 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 o 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 lter for Western
14. to rapidly characterize plasmid clones study structural mutations and examine translational signals Two basic approaches to in vitro protein synthesis are available 1 systems programmed with RNA translation systems or 2 systems programmed with DNA coupled 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 o ers several Rabbit Reticulocyte Lysate and Wheat Germ Extract Systems All are reliable convenient and easy to use systems to initiate translation and produce full size polypeptide products 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 Flexi Rabbit Reticulocyte Lysate is recommended where optimization of translation of particular RNAs through adjustments to salt and DTT concentrations is required Wheat Germ Extract is recommended for translation of RNA preparations containing low concentrations 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 usi
15. 30 1767 1796 pUC M13 reverse primer 17mer 1833 1817 pUC M13 reverse primer 22mer 1838 1817 lactamase gene Ampr 3838 2978 SP6 RNA polymerase promoter primer 4731 1 SP6 RNA polymerase promoter 4731 3 28 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 11 B Luciferase SP6 T7 Control DNAs continued ori Luciferase T7 Control DNA 4331bp Ampr XmnI 2632 luc dA dT 30 SacI 1767 T7 Initiation 1 T7 Promoter BamHI 44 NotI 22 HindIII 11 1916VA04_6A Figure 4 Luciferase T7 Control DNA circle map and sequence reference points Additional description Ampr lactamase gene resistant to ampicillin ori origin of plasmid replication Sequence reference points T7 RNA polymerase initiation 1 GLPrimer2 52 74 Luciferase gene 51 1700 Poly A dA 30 1770 1799 lactamase gene Ampr 2444 3304 T7 RNA polymerase promoter 4315 3 T7 RNA polymerase promoter primer 4315 3 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 29 www promega com TM045 Revised 12 13 11 C Related Products The in vitro synthesis of proteins is a popular method in biological research Among other applications translation systems are used
16. 6 1 www promega com TM045 Revised 12 13 2 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 1 Description The TNT Quick Coupled Transcription Translation Systems a b c are convenient single tube coupled transcription translation reactions for eukaryotic in vitro translation The original TNT Coupled Reticulocyte Lysate Systems simpli ed the process and reduced the time required to obtain in vitro translation results compared with standard rabbit reticulocyte lysate systems 1 Standard rabbit reticulocyte systems commonly use RNA synthesized in vitro from SP6 T3 or T7 RNA polymerase 1 The TNT Quick Coupled Transcription Translation System further simpli es the process by combining 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 produces signi cantly more protein two to sixfold in a 60 to 90 minute reaction than a standard in vitro rabbit reticulocyte lysate reaction using RNA templates The TNT Quick Coupled Transcription Translation System is available in two con gurations for transcription and translation of genes cloned downstream from either the T7 or SP6 RNA polymerase promoters To use these systems
17. C for 60 90 minutes 5 Analyze the results of translation A protocol for Western Blot analysis is provided in Section 7 A Procedures for determining radiolabel incorporation Section 7 B and SDS PAGE analysis of translation products Section 7 C are provided If using FluoroTect GreenLys tRNA see Section 7 D for Transcend tRNA reactions see Section 7 E 4 B General Protocol for TNT T7 Quick Coupled Transcription Translation Reactions Using PCR Generated DNA Materials to Be Supplied by the User Nuclease Free Water Cat P1193 Radiolabeled amino acid for radioactive detection Note 4 Section 4 C or Transcend tRNA Cat L5061 or Transcend Colorimetric Cat L5070 or Chemiluminescent Cat L5080 Translation Detection System for non radioactive detection or FluoroTect GreenLys in vitro Translation Labeling System for uorescent 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 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 adding all of the components gently mix by pipetting If necessary centrifuge brie y 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 4 C
18. P64 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 Puri cation System or by phenol chloroform extraction followed by ethanol precipitation before use in the TNT Quick reaction 3 Plasmid DNA can be puri ed using the Wizard Plus SV Minipreps DNA Puri cation System or the PureYield Plasmid Midiprep System DNA prepared by the standard alkaline lysis method described by Sambrook Fritsch and Maniatis 14 is also su ciently clean for use in the TNT Quick Coupled Transcription Translation System For most constructs optimal results are obtained when 1 g of plasmid DNA template is used However we have used 0 2 2 0 g of DNA template and obtained satisfactory levels of translation The use of more than 1 g of plasmid does not necessarily increase the amount of protein produced 4 If linearizing plasmid DNA for use with the T7 System avoid the use of restriction enzymes that yield 3 overhangs PstI KpnI SacI SacII BstXI NsiI ApaI and AatII as aberrant transcription products can be produced 15 If no alternative enzyme is available the 3 overhang can be removed by adding T4 DNA polymerase Note If you are using a linearized plasmid as a template include 1 l of the T7 TN
19. Revised 12 13 TM045 T E C H N I C A L M A N U A L TNT Quick Coupled Transcription Translation Systems Instruc ons for use of Products L1170 L1171 L2080 and L2081 All technical literature is available at www promega com protocols Visit the web site to verify that you are using the most current version of this Technical Manual E mail Promega Technical Services if you have questions on use of this system techserv promega com TNT Quick Coupled Transcription Translation Systems 1 Description 2 2 Product Components and Storage Conditions 5 3 General Considerations 6 3 A DNA Template Considerations 6 3 B Creating a Ribonuclease Free Environment 8 3 C Handling of Lysate 8 4 Translation Procedure
20. T PCR Enhancer in each 50 l reaction 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 rst AUG encountered 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 8 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 3 A DNA Template Considerations continued PCR Generated DNA Templates 1 Because PCR DNA templates are usually much smaller than plasmid templates the amount of DNA necessary for optimal expression is often less than for inserts cloned into plasmid vectors e g for a 500bp PCR product use 100 800ng for each 50 l TNT Quick reaction Note For coupled transcription translation from PCR generated templates Promega o ers TNT T7 Quick for PCR DNA Cat L5540 2 PCR products 5 7 l can be used directly from the ampli cation reaction Note If you are using a PCR generated template include 1 l of the T7 TNT PCR Enhancer in each 50 l reaction 3 B Creating a Ribonuclease Free Environment To reduce
21. actions Includes 200 l TNT Quick Master Mix 5 g SP6 or T7 Luciferase Control DNA 0 5 g l 100 l T7 TNT PCR Enhancer L1171 only 50 l Methionine 1mM Storage and Stability Store all components at 70 C Product components are sensitive to CO2 avoid prolonged exposure frequent temperature uctuations and multiple freeze thaw cycles which can adversely a ect stability activity and performance Luciferase Assay Reagent LAR is stable for at least 12 months if stored and handled properly Note See Note 5 Section 4 C for details on how to refreeze the lysate Note that the systems are shipped in foil packaging because the system is sensitive to carbon dioxide released from dry ice If storing the system in a freezer containing dry ice keep system components 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 signi cant 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 two times 3 General Considerations 3 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 we recommend that approximately 11bp be present upstream of the T7 RNA polymerase promote
22. actions L4611 30 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 11 C Related Products continued TNT Coupled Wheat Germ Extract Systems Product Size Cat TNT SP6 High Yield Wheat Germ Protein Expression System 4 300 l L3260 1 300 l L3261 TNT T3 Coupled Wheat Germ Extract System 40 reactions L4120 TNT SP6 Coupled Wheat Germ Extract System 40 reactions L4130 TNT T7 Coupled Wheat Germ Extract System 40 reactions L4140 TNT T7 SP6 Coupled Wheat Germ Extract System 40 reactions L5030 TNT T7 T3 Coupled Wheat Germ Extract System 40 reactions L5040 Rabbit Reticulocyte Lysate Product Size Cat Rabbit Reticulocyte Lysate Nuclease Treated 5 200 l L4960 Rabbit Reticulocyte Lysate Untreated 1ml L4151 Bulk Rabbit Reticulocyte Lysate is available from Promega Flexi Rabbit Reticulocyte Lysate System Product Size Cat Flexi Rabbit Reticulocyte Lysate System 5 200 l L4540 Bulk Flexi Rabbit Reticulocyte Lysate is available from Promega Wheat Germ Extract Product Size Cat Wheat Germ Extract 5 200 l L4380 Rabbit Reticulocyte Lysate Wheat Germ Extract Combination System Product Size Cat Rabbit Reticulocyte Wheat Germ Extract Combination System 12 reactions each L4330 Promega Corpora on 2800 Woo
23. ased uorescence scanning device Note The use of gel systems other than Tris Glycine may cause di erent migration patterns for the expressed and background bands Denaturing Gel Analysis 1 Once the translation reaction is complete or at any desired time point remove a 5 l aliquot and add it to 20 l of 1X SDS gel loading bu er Store the remainder of the translation reaction at 20 C The FluoroTect tRNA uorophore is sensitive to extreme heating If heating to denature the proteins do not exceed 70 C for more than 2 3 minutes 2 Load the sample from Step 1 on an SDS PAGE gel 3 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 to the bottom of the gel Fluorescent Detection Materials to Be Supplied by the User Fluorescent Imaging Instrument i e FluorImager SI or FluorImager 595 Molecular Dynamics both with a 499 argon laser the Typhoon 8600 Molecular Dynamics with a 532nm excitation or the FMBIO II Hitachi with a 505 channel Note The Storm instrument Molecular Dynamics is not recommended for use with the FluoroTect System due to reduced sensitivity After electrophoresis is completed immediately place the gel in water then complete uorescent scanning Use gloves when handling the gels Notes
24. beled with the uorophore BODIPY FL to incorporate uorescently labeled lysine residues into the in vitro translation product For more information on the FluoroTect System request Technical Bulletin TB285 Note Technical Manuals and Bulletins are available online at www promega com tbs or by request from Technical Services Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 9 www promega com TM045 Revised 12 13 4 A General Protocol for TNT Quick Coupled Transcription Translation Reactions Using Plasmid DNA Materials to Be Supplied by the User Nuclease Free Water Cat P1193 Radiolabeled amino acid for radioactive detection Note 4 Section 4 C or Transcend tRNA Cat L5061 or Transcend Colorimetric Cat L5070 or Chemiluminescent Cat L5080 Translation Detection System for non radioactive detection or FluoroTect GreenLys in vitro Translation Labeling System for uorescent 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 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 adding all of the components gently mix by pipetting If necessary centrifuge brie
25. blotting proceed to Step 7 5 Place the polyacrylamide gel in a plastic box and cover the gel with xing solution as prepared in Section 11 A for 30 minutes Agitate slowly on an orbital shaker Pour o the xing solution Proceed to Step 6 gel drying prior to lm exposure Optional Labeled protein bands in gels may be visualized by autoradiography or uorography Fluorography dramatically increases the sensitivity of detection of 35S 14C and 3H labeled proteins and is recommended for the analysis of in vitro translation products The increased detection sensitivity of uorography 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 lm Commercial reagents such as Amplify Reagent GE Healthcare Bio sciences can be used for uorographic enhancement of signal Alternatively the xed gel can be exposed to a phosphorimaging screen These systems provide greater sensitivity greater speed and the ability to quantitate the radioactive bands 6 Dry the gel before exposure to lm as follows Soak the gel in 10 glycerol for 5 minutes to prevent the gel from cracking during drying Place the gel on a sheet of Whatman 3MM lter 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
26. cend tRNA and FluoroTect GreenLys tRNA can be increased 1 4 l to allow more sensitive detection of proteins that contain few lysines or are poorly expressed Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 13 www promega com TM045 Revised 12 13 5 Positive Control Translation Reactions Using Luciferase The assay for re y luciferase activity is extremely sensitive rapid and easy to perform 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 modi cation for enzymatic activity The Luciferase Assay System is a substantial improvement over conventional methods in both sensitivity and simplicity 17 The control reaction can be performed with or without the addition of radiolabeled amino acids 5 A Radioactive Luciferase Control Reaction 1 The following example contains 35S methionine TNT Quick Master Mix see Note 3 Section 4 C 40 l 35S methionine 1 000Ci mmol at 10mCi ml see Note 4 Section 4 C 2 l Appropriate Luciferase Control DNA 0 5 g l see Section 11 B 2 l Nuclease Free Water to a nal volume of 50 l 2 Incubate the reaction at 30 C for 60 90 minutes see Note 3 Section 4 C 3 Analyze the results of translation by measur
27. d 12 13 18 Andrews D 1987 Assaying protein translocation across the endoplasmic reticulum membrane Promega Notes 11 1 4 19 Towbin H et al 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets Procedure and some applications Proc Natl Acad Sci USA 76 4350 4 20 Burnette W N 1981 Western blotting Electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodi ed nitrocellulose and radiographic detection with antibody and radioiodinated protein A Anal Biochem 112 195 203 21 Bittner M et al 1980 Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxy methyl cellulose or nitrocellulose sheets Anal Biochem 102 459 71 22 Towbin H and Gordon J 1984 Immunoblotting and dot immunobinding current status and outlook J Immunol Meth 72 313 40 23 Bers G and Gar n D 1985 Protein and nucleic acid blotting and immuno biochemical detection BioTechniques 3 276 88 24 Hicks D et al 1986 Immobilon PVDF Transfer Membrane A new membrane substrate for Western blotting of proteins BioTechniques 4 272 25 Protocols and Applications Guide Online Edition 2004 2006 Promega Corporation 26 Hurst R et al 1996 The TNT T7 Quick Coupled Transcription Translation System Promega Notes 58 8 11 27 Kozak M 1986 Point mutations de ne a seq
28. d add it to 15 l of SDS sample bu er 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 10 minutes at 70 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 found in the Protocols and Applications Guide online edition 25 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 o the bottom of the gel Note If a gene product is weakly expressed or contains few lysines up to 2 l 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 background on the blot Electroblotting of Proteins to Membrane For colorimetric detection see Section 5 C of the Transcend Non Radioactive Translation Detection Systems Technical Bulletin TB182 The translation products can be blotted from the SDS polyacrylamide gel to in decreasing order of preference PVDF nitrocellulose or an
29. ds Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 31 www promega com TM045 Revised 12 13 E coli S30 Extract Systems Product Size Cat E coli S30 Extract System for Linear Templates 30 reactions L1030 E coli S30 Extract System for Circular DNA 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 Non Radioactive Translation Detection System 30 reactions L5070 Transcend Chemiluminescent Non Radioactive Translation Detection System 30 reactions L5080 Transcend Biotinylated tRNA 30 l L5061 FluoroTect GreenLys in vitro Translation Labeling System Product Size Cat FluoroTect GreenLys in vitro Translation Labeling System 40 reactions L5001 Canine Pancreatic Microsomal Membranes Product Size Cat Canine Pancreatic Microsomal Membranes 50 l Y4041 Protein Protein Interactions Product Size Cat MagneGST Pull Down System 80 reactions V8870 Plasmid Puri cation Product Size Cat PureYield Plasmid Midiprep System 25 preps A2492 100 preps A2495 32 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com a U S Pat Nos 5 641
30. e with Canine Microsomal Membranes Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 15 www promega com TM045 Revised 12 13 Notes 1 We do not recommend using Canine Microsomal Membranes when using SP6 TNT Quick Coupled Transcription Translation Systems because SP6 polymerase is sensitive to salts Transcription may be inhibited as much as 70 by the presence of Canine Microsomal Membranes in the reaction 2 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 e ciency of processing using membranes may vary Thus reaction parameters may need to be altered to suit individual requirements In general increasing the amount of membranes in the reaction increases the proportion of polypeptides processed but reduces the total amount of polypeptides synthesized 3 For reactions using the TNT Quick Coupled Transcription Translation System the Canine Microsomal Membranes will inhibit transcription We do not recommend exceeding 1 8 l of Canine Microsomal Membranes Transcription Translation may be inhibited by as much as 50 with 0 6 l of Canine Microsomal Membranes 4 The amount of protein produced in TNT Quick reactions using Canine Pancreatic Microsomal Membranes will be less than the amount produced in TNT Quick reactio
31. eaction 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 proteins and is often used to verify the in vivo results obtained from yeast two hybrid experiments Promega o ers the MagneGST Pull Down System Cat V8870 for GST pull down experiments 2 Product Components and Storage Conditions P R O D U C T C AT TT T7 Quick Coupled Transcrip on Transla on System L1170 TT SP6 Quick Coupled Transcrip on Transla on System L2080 Each system contains su cient reagents to perform approximately 40 50 l translation reactions Includes 1 6ml TNT Quick Master Mix 8 200 l 5 g SP6 or T7 Luciferase Control DNA 0 5 g l 100 l T7 TNT PCR Enhancer L1170 only 50 l Methionine 1mM 250 l Luciferase Assay Reagent 1 25ml Nuclease Free Water 6 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 2 Product Components and Storage Conditions continued P R O D U C T C AT TT T7 Quick Coupled Transcrip on Transla on System Trial Size L1171 TT SP6 Quick Coupled Transcrip on Transla on System Trial Size L2081 Each system contains su cient reagents to perform approximately 5 50 l translation re
32. ed protein Hemoglobin present in the rabbit reticulocyte lysate will bind to the nickel and co elute with the polyhistidine tagged protein Use the MagZ Protein Puri cation System Cat V8830 or an alternate puri cation tag to isolate the protein from the TNT lysate and avoid this problem Smearing on the gel Gel not clean Gel must be washed before placing onto lm Once gel electrophoresis is complete soak the gel in either a standard Coomassie destaining solution 50 methanol 7 5 glacial acetic acid or in water for 15 30 minutes prior to drying Too much protein loaded on the gel Check the amount of sample loaded on the gel and the amount of loading bu er Too much protein loaded can cause smearing Acrylamide concentration in the gel is too low Acrylamide concentration can be increased to 12 Sample contains ethanol which can cause gel smearing High background levels when Primary antibody concentration too high Increase the dilution performing Western Blots of the primary antibody 24 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 10 References 1 Pelham H R B and Jackson R J 1976 An e cient mRNA dependent translation system from reticulocyte lysates Eur J Biochem 67 247 56 2 Bibliography of References Using the TNT Coupled Transcriptio
33. he gel Proteolysis of translation product Add protease inhibitors such as 2 macroglobulin leupeptin or chymostatin 0 5 1 g ml More than one peptide is translated from the template Leaky scanning for translation initiation can result in translation initiating at internal methionines Optimizing the Mg2 or K concentration can increase delity 26 The 35S methionine used is not translational grade or beyond its expiration date There are reports of a 42kDa band with some grades of 35S methionine 16 We recommend Perkin Elmer EasyTag L 35S methionine Perkin Elmer Cat NEG709A to avoid this 42kDa band Globin may appear on the autoradiogram or stained gel It appears as a broad band migrating at 10 15kDa Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 23 www promega com TM045 Revised 12 13 Symptoms Causes and Comments Unexpected bands present on the gel Aminoacyl tRNAs may produce background bands 25kDa continued Add RNase A to the lysate reaction after completion to a nal concentration of 0 2mg ml Incubate for 5 minutes at 30 C Oxidized mercaptoethanol is present or not enough SDS in the loading bu er Use a loading bu er that contains 2 SDS and 100mM DTT Unexpected bands present when A nickel based resin is used to purify polyhistidine tagged proteins isolating polyhistidine tagg
34. ine PerkinElmer EasyTag L 35S methionine PerkinElmer Cat NEG709A which does not cause the background labeling of the rabbit reticulocyte lysate 42kDa protein Background labeling of the 42kDa protein can occur using other grades of label 16 Between 10 40 Ci 1 4 l of 35S methionine can be added to the TNT Quick reactions depending upon the balance between labeling e ciency and cost For gene constructs that express well and contain several methionines the 10 Ci level 1 l is su cient 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 1 g of plasmid DNA template is used We recommend using 0 2 2 0 g of plasmid DNA in TNT Quick reactions The use of more than 1 g of plasmid does not necessarily increase the amount of protein produced 7 Avoid adding calcium to the transcription translation reaction Calcium may reactivate 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 9 The level of added Trans
35. ing direct incorporation of radiolabel Section 7 B and or gel analy sis of translation products Section 7 C 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 5 B Non Radioactive Luciferase Control Reaction 1 The following example contains Methionine TNT Quick Master Mix see Note 3 Section 4 C 40 l Methionine 1mM 1 l Appropriate Luciferase Control DNA 0 5 g l see Section 11 B 2 l Nuclease Free Water to a nal volume of 50 l 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 8 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 little loss of luciferase activity 14 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 6 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
36. lation products Section 7 C are provided If using FluoroTect GreenLys tRNA see Section 7 D for Transcend tRNA reactions see Section 7 E 12 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 4 C Notes 1 PCR generated templates can be used directly from the ampli cation reaction We recommend using 2 5 5 l from the ampli cation reaction but up to 7 l can be used in a 50 l reaction For PCR generated DNA that has been puri ed following ampli cation we recommend using 100 800ng of the puri ed product for each reaction 2 We recommend using 1 l of the T7 TNT PCR Enhancer in a 50 l reaction to increase transcription translation when using PCR generated DNA linear plasmid or viral enhanced plasmids 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 di er in the Mg2 and K concentrations required for optimal expression in the coupled reaction For example some viral leaders will increase translation e ciency and delity 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 2 l of the T7 TNT PCR Enhancer 4 We recommend using a grade of 35S methion
37. m temperature T7 TNT PCR Enhancer 0 5M KCl 12 5mM Mg OAc 2 In nanopure water TBST 50mM Tris HCl pH7 4 150mM NaCl 0 1 Tween 20 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 27 www promega com TM045 Revised 12 13 11 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 ori Luciferase SP6 Control DNA 4747bp Ampr luc XmnI 3651 SP6 Initiation 1 SP6 Promoter HindIII 8 NotI 21 BamHI 41 SacI 1764 XmnI 1804 dA dT 30 1917VA04_6A Figure 3 Luciferase SP6 Control DNA circle map and sequence reference points Additional description Ampr lactamase gene resistant to ampicillin ori origin of plasmid replication Sequence reference points SP6 RNA polymerase initiation 1 GLPrimer2 49 71 Luciferase gene 48 1700 Poly A dA
38. n Translation Systems BL001 1996 Promega Corporation 3 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 4 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 5 Sharp T V Witzel J E and Jagus R 1997 Homologous regions of the alpha subunit of eukaryotic translational initiation factor 2 eIF2alpha and the vaccinia virus K3L gene product interact with the same domain within the dsRNA activated protein kinase PKR Eur J Biochem 250 85 91 6 Jagus R and Beckler G S 1998 Overview of eukaryotic in vitro translation and expression systems Current Protocols in Cell Biology Bonifacirro et al eds John Wiley amp Sons Inc 11 1 1 11 1 13 7 Cleveland D L and Ihle J H 1995 Contenders in FasL TNF death signaling Cell 81 479 82 8 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 9 Chien 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 7 10 FluoroTect GreenLys in vitro Translation Labeling Sys
39. ng either TNT Coupled Reticulocyte Lysate Systems Wheat Germ Extract Systems or E coli S30 Extract Systems 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 consensus context A GCCAUGG 27 in the absence of inhibiting secondary structure The template DNA to be expressed in the S30 Systems must contain E coli promoter sequences or a phage T7 promoter sequence and prokaryotic ribosome 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 40 reactions L4600 TNT T7 Coupled Reticulocyte Lysate System 40 reactions L4610 TNT T3 Coupled Reticulocyte Lysate System 40 reactions L4950 TNT T7 T3 Coupled Reticulocyte Lysate System 40 reactions L5010 TNT T7 SP6 Coupled Reticulocyte Lysate System 40 reactions L5020 TNT T7 Quick for PCR DNA 40 reactions L5540 TNT SP6 Coupled Reticulocyte Lysate System Trial Size 8 reactions L4601 TNT T7 Coupled Reticulocyte Lysate System Trial Size 8 re
40. ns alone Depending on the construct used protein synthesis e ciency can be expected to drop between 10 50 in the presence of Microsomal Membranes 5 In some cases it is di cult to determine if e cient processing or glycosylation has occurred by gel analysis alone Other assays such as various protection assays 18 may be required to determine if processing events have taken place 7 Post Translational Analysis Materials to Be Supplied by the User Solution compositions are provided in Section 11 A 1M NaOH 2 H2O2 25 TCA 2 casamino acids Difco brand Vitamin Assay Grade 5 TCA Whatman GF A glass ber lter Whatman Cat 1820 021 acetone Whatman 3MM lter paper 30 acrylamide solution separating gel 4X bu er stacking gel 4X bu er SDS sample bu er SDS polyacrylamide gels 50mM DTT Blot Quali ed BSA Cat W3841 PVDF membrane iBlot SP antibody TBST bu er 16 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 7 A Western Blot Analysis 1 Add 1 l of the standard unlabeled translation reaction to 19 l of 1X SDS loading dye with 50mM DTT Note Include a no template control on the gel to identify background bands 2 Incubate at 95 C for 5 minutes Centrifuge
41. o a linear relationship between luciferase concentration and cpm still can be produced by calculating the square root of measured counts per minute cpm minus background cpm i e sample background 1 2 To measure background cpm use water or Luciferase Assay Reagent as a blank Use the same protocol as luciferase assays using a luminometer Section 7 B 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 scintillant because it will inactivate luciferase Alternatively place the sample in a microcentrifuge tube and then place the tube in the scintillation vial To ensure consistency when working with multiple samples place each micro centrifuge 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 22 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega c
42. om 9 Troubleshooting For questions not addressed here please contact your local Promega Branch O ce or Distributor Contact information available at www promega com E mail techserv promega com Symptoms Causes and Comments The control reaction produces no luciferase Loss of reaction component s activity The lysate should not be used after more than two freeze thaw cycles Do not use reagents after the expiration date Ethanol or salt present in the reaction may inhibit translation Low translation e ciency Certain gene constructs may require di erent Mg2 and K concentrations for optimal expression Add 1 3 l of the T7 TNT PCR Enhancer Calcium is present in the translation reaction Avoid adding calcium to the translation reaction Calcium may reactivate the micrococcal nuclease used used to destroy endogenous mRNA in the lysate and result in degradation of the DNA or mRNA template Ethanol present in the translation reaction Residual ethanol should be removed from template DNA preparations and amino acids before they are added to the translation reaction Incubation of the reaction at 37 C causes decreased protein synthesis Incubate the reaction at 30 C the optimal temperature Unexpected bands present Denaturing temperature too high Denature sample at a lower at higher molecular weights temperature e g 60 80 C for 10 15 minutes or bands stuck in stacking gel Unexpected bands present on t
43. other membrane using any standard apparatus and protocol including semi dry systems Detailed procedures for electrophoretic blotting are usually included with commercial devices We routinely 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 bu er Instructions for chemiluminescent detection of products are found in Section 5 D of the Transcend Non Radioactive Translation Detection Systems Technical Bulletin TB182 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 21 www promega com TM045 Revised 12 13 8 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 measurements 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 u
44. r a total of six washes 16 Proceed to the detection method appropriate for your secondary antibody If using FluoroTect GreenLys tRNA see Section 7 D for Transcend tRNA reactions see Section 7 E Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 17 www promega com TM045 Revised 12 13 7 B Determination of Percent Incorporation of Radioactive Label 1 After the 50 l translation reaction is complete remove 2 l from the reaction and add it to 98 l of 1M NaOH 2 H2O2 2 Vortex brie y and incubate at 37 C for 10 minutes 3 At the end of the incubation add 900 l of ice cold 25 TCA 2 casamino acids to precipitate the translation product Incubate on ice for 30 minutes 4 Wet a Whatman GF A glass ber lter with a small amount of ice cold 5 TCA Collect the precipitated translation product by vacuum ltering 250 l of the TCA reaction mix Rinse the lter 3 times with 1 3ml of ice cold 5 TCA Rinse once with 1 3ml of acetone Allow the lter to dry at room temperature or under a heat lamp for at least 10 minutes 5 For determination of 35S incorporation put the lter in the appropriate scintillation cocktail invert to mix and count in a liquid scintillation counter 6 To determine total counts present in the reaction spot a 5 l aliquot of the TCA reaction mix directly onto a lter Dr
45. r for e cient 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 11 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 transcription e ciencies Note For coupled transcription translation from PCR generated templates Promega o ers TNT T7 Quick for PCR DNA Cat L5540 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 7 www promega com TM045 Revised 12 13 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 region because this can impair translation e ciency 12 3 We have observed enhanced translation of proteins when using DNA constructs 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 13 For example we have observed a two to vefold increase in the production of luciferase when the gene is cloned into the pS
46. ree in USA 800 356 9526 608 274 4330 Fax 608 277 2516 3 www promega com TM045 Revised 12 13 TNT Coupled Reticulocyte Lysate System TNT Rabbit Reticulocyte Lysate Add TNT Reaction Buffer Add TNT RNA Polymerase Add Amino Acid Mixture Minus Methionine Add RNasin Ribonuclease Inhibitor TNT Quick Coupled Transcription Translation System TNT Quick Master Mix Add label of choice Add DNA template and Nuclease Free Water Incubate at 30 C for 60 90 minutes Separate translation products by SDS PAGE Detect 1537MB11_2A Figure 1 Comparison of the TNT Coupled Reticulocyte Lysate System and the TNT Quick Coupled Transcription Translation System protocols 4 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 1 Description continued 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 Truncation mutation analysis e g the Protein Truncation Test PTT 2 Drug screening a ecting translation rates Mutation and detection analysis i e enzyme kinetics Protein protein interactions using GST pull downs
47. sed for quantitation There is usually insu cient 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 limits 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 instrument operator s manual for general operating instructions 8 A Using a Luminometer 1 Dispense 50 l 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 su cient light is produced 3 Add 2 5 l of cell lysate to a luminometer tube containing the Luciferase Assay Reagent Mix by pipetting 2 3 times or vortex brie y 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 8 B Using a Scintillation Counter Ideally the coincidence circuit of the scintillation counter should be turned o 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
48. tem Technical Bulletin TB285 Promega Corporation 11 Beckler G et al 2000 A new TNT System for enhanced expression of PCR DNA Promega Notes 74 10 13 12 Frances V Morle F and Godet J 1992 Identi cation of two critical base pairings in 5 untranslated regions a ecting translation e ciency of synthetic uncapped globin mRNAs Biochim Biophys Acta 1130 29 37 13 Jackson R J and Standart N 1990 Do the poly A tail and 3 untranslated region control mRNA translation Cell 62 15 24 14 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY 15 Schenborn E T and Mierendorf R C 1985 A novel transcription property of SP6 and T7 RNA polymerases Dependence on template structure Nucl Acids Res 13 6223 36 16 Jackson R J and Hunt T 1983 Preparation and use of nuclease treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA Meth Enzymol 96 50 74 17 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 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 25 www promega com TM045 Revise
49. the chance of RNase contamination gloves should be worn when setting up experiments and micro centrifuge 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 3 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 4 C Do not freeze thaw the Master Mix more than two times 4 Translation Procedure The following is a general guideline for setting up a transcription translation reaction Also provided are examples of standard reactions using 35S methionine radioactive Transcend Non Radioactive Detection System colorimetric or chemiluminescent or FluoroTect GreenLys Systems uorescent Using the Transcend Systems biotinylated lysine residues are incorporated 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 Technical Bulletin TB182 The FluoroTect System uses a charged lysine tRNA la
50. tinylated lysine e g Transcend Biotinylated tRNA or is uorescently tagged e g FluoroTect GreenLys System BODIPY FL labeled tRNA Cat L5001 and combined with a GST tagged protein The biotinylated protein is detected by methods similar to those used in Western blotting 8 9 The uorescently tagged protein can be detected from within the gel 10 For a complete list of references for these and other applications see reference 6 or visit the Promega Technical Resource Center citations at www promega com citations Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 5 www promega com TM045 Revised 12 13 35S Gene 1 TNT System Protein 1 GST Gene 1 Purify on Affinity Column Express in E coli GST Protein 2 Western W Wash E Eluate M Marker Autoradiography W E M W E M 2598MA03_9A Figure 2 The study of protein protein interactions using the TNT Systems 6 This schematic shows translation of one protein with radioactive 35S methionine in a TNT System reaction Large amounts of the suspected partner protein are expressed and puri ed A fusion tag most commonly GST is incorporated into this second protein to facilitate puri cation 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 r
51. uence anking the AUG initiator codon that modulates translation by eukaryotic ribosomes Cell 44 283 92 26 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM045 Revised 12 13 www promega com 11 Appendix 11 A Composition of Bu ers and Solutions acrylamide solution 30 37 5 1 30g acrylamide 0 8g bisacrylamide Add water to a nal volume of 100ml Store at 4 C xing solution 50 methanol 10 glacial acetic acid 40 water 1X SDS gel loading bu er 50mM Tris HCl pH 6 8 100mM dithiothreitol 2 SDS 0 1 bromophenol blue 10 glycerol 1X SDS gel loading bu er lacking dithiothreitol can be stored at room temperature Dithiothreitol should be added from a 1M stock just before the bu er is used SDS polyacrylamide running 10X bu er 30g Tris base 144g glycine 100ml 10 SDS Add deionized water to a nal volume of 1 liter Store at room temperature separating gel 4X bu er 18 17g Tris base 4ml 10 SDS Bring the volume to approximately 80ml with deion ized water Adjust to pH 8 8 with 12N HCl and add deionized water to a nal volume of 100ml Store at room temperature stacking gel 4X bu er 6 06g Tris base 4ml 10 SDS Bring the volume to approximately 80ml with deion ized water Adjust to pH 6 8 with 12N HCl and add deionized water to a nal volume of 100ml Store at roo
52. y the lter for 10 minutes Count in a liquid scintillation counter as in Step 5 7 To determine background counts remove 2 l from a 50 l translation reaction containing no DNA and proceed as described in Steps 1 5 8 Perform the following calculation to determine percent incorporation cpm of washed lter Step 5 100 percent incorporation cpm of unwashed lter Step 6 50 9 Perform the following calculation to determine the fold stimulation over background cpm of washed lter Step 5 fold stimulation cpm of no DNA control reaction lter Step 7 7 C Denaturing Gel Analysis of Radioactively Labeled Translation Products Precast polyacrylamide gels are available from a number of manufacturers For protein analysis Invitrogen Corporation and Bio Rad Laboratories Inc o er a variety of precast mini gels which are compatible with their vertical electro phoresis and blotter systems These companies o er Tris Glycine Tricine and Bis Tris gels for resolution of proteins under di erent conditions and over a broad spectrum of protein sizes The Invitrogen Novex 4 20 Tris Glycine gradient gels Cat EC6025BOX or EC60355BOX and the Bio Rad Ready Gel 4 20 Tris Glycine Gel 10 well 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 50 l translation
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