TNT® Quick Coupled Transcription/Translation Systems
Contents
1. For your pr aten pur ification needs please see the related 6 Cotranslational Processing Using Canine Pancreatic products section of this Technical Manual or visit Microsomal Membranes 14 www promega com for a complete listing of protein purification A General Protocol for Translation with Microsomal Membranes 14 products offered by Promega 7 Post Translational Analysis A Determination of Percent Incorporation of Radioactive Label B Denaturing Gel Analysis of Radioactively Labeled Translation Products oiiaee a aSr 16 C Denaturing Gel Analysis of Translation Products Labeled with the FluoroTect Green in vitro Translation Labeling System D Denaturing Gel Analysis of Translation Products Labeled with the Transcend Non Radioactive Translation Detection Systems 19 8 Positive Control Luciferase Assays A Using a Luminometer B Using a Scintillation Counter isssssssssssessisioessisssssssecsesessssvoosseissessaissecesssiveoes 9 Trot leshootinig sis ccsscsisscissstscestsvasssssazencsbesssvasssceestvtinseajesavisceabenavisdzasissstelveets 21 10 References aami ahai iE liven E cena 23 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Rart M0415 Revised 3 09 Page 1 Oo Promega 11 Appendix A Composition of Buffers an2. S 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 5S 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 biotinylated lysine e g Transcend Biotinylated tRNA or is fluorescently tagged e g FluoroTect Green 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 fluorescently 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 Gene 1 GST Gene 1 Express in E coli Purify on Affinity Column TnT System GST Protein 2 Protei
3. 0 5pg pl see Note 6 Section 4 C ul 2pl 2pl Transcend Biotin Lysyl tRNA see Note 9 Section 4 C 1 2pl FluoroTect Greeny tRNA see Note 9 Section 4 C aes 1 2 Nuclease Free Water to a final volume of 50pl 50pl 50pl Note Small scale reactions may be performed by reducing the recommended volumes proportionally Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Printed in USA Page 8 Revised 3 09 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 9 Oo Promega Oo Promega 4 A General Protocol for TNT Quick Coupled Transcription Translation Reactions Using Plasmid DNA continued 4 Incubate the reaction at 30 C for 60 90 minutes 5 Analyze the results of translation Procedures for determination of radiolabel incorporation Section 7 A and SDS PAGE analysis of translation products Section 7 B are provided If using FluoroTect Greenyy tRNA see Section 7 C for Transcend tRNA reactions see Section 7 D 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 e Radiolabele
4. 6 Revised 3 09 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 7 Oo Promega 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 501 TNT Quick reaction Note For coupled transcription translation from PCR generated templates Promega offers TNT T7 Quick for PCR DNA Cat L5540 2 PCR products 5 71 can be used directly from the amplification reaction Note If you are using a PCR generated template include 1pl of the T7 TNT PCR Enhancer in each 50pl reaction 3 B Creating a Ribonuclease Free Environment To reduce the chance of RNase contamination gloves should be worn when setting 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 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 refroze
5. Eur J Biochem 67 247 56 2 Bibliography of References Using the TNT Coupled Transcription 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 elF2alpha 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 C
6. The storage buffer 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 20pl S methionine 1 000Ci mmol at 10mCi ml see Note 4 Section 4 C 2 0p1 plasmid DNA 0 5pg pl 0 5pl Canine Pancreatic Microsomal Membranes see Note 1 below 0 3 1 8p1 Nuclease Free Water to a final volume of 25pl 3 Incubate at 30 C for 60 90 minutes 4 Analyze the results of translation and processing Procedures for incorporation assays Section 7 A and SDS PAGE analysis of translation products Section 7 B are provided Note TNT Quick Coupled Transcription Translation Systems are not tested for performance with Canine Microsomal Membranes 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 Oo Promega Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 gt www promega com Part TM045 Printed in USA Page 14 Revised 3 09 2 The amount of Canine Microsomal Membranes used in the reaction may need to be titrated While these reaction conditio
7. a 1p aliquot and add it to 151 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 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 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 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 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 1B182 The translation products can be blotted from the SDS polyacrylamide gel to in decreasing order of preference PVDF nitrocellulose or another memb
8. by placing the gel on a laser based fluorescence scanning device Note The use of gel systems other than Tris Glycine may cause different 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 5pl aliquot and add it to 2041 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 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 e Fluorescent Imaging Instrument i e Fluorlmager SI or Fluorlmager 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 fluorescent scanning Use gloves when handling the g
9. 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 Phone 608 274 4330 Fax 608 277 2516 gt www promega com Part TM045 Printed in USA Page 24 Revised 3 09 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 3 09 Part TM045 Page 25 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 SP6 Promoter _ SP6 Initiation 1 Hindili 8 Not 21 BamHI 41 i Bua Luciferase SP6 Control DNA 4747bp ay N Dn 1917VA04_6A Figure 3 Luciferase SP6 Control DNA circle map and sequence reference points Additi
10. 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 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 unmodified 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 diazobenzyloxymethy 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 Garfin D 1985 Protein and nucleic acid blotting and immuno biochemical detection BioTechniques 3 276 88 24 Hicks D et al 1986 Immobilon PVDF Tr
11. A 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 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 14600 TNT T7 Coupled Reticulocyte Lysate System 40 reactions 14610 TNT T3 Coupled Reticulocyte Lysate System 40 reactions 14950 TNT T7 T3 Coupled Reticulocyte Lysate System 40 reactions 15010 TNT T7 SP6 Coupled Reticulocyte Lysate System 40 r
12. 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 0 001 pg 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 50pl 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 5pl 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 8 B Using a Scintillation Counter Ideally the coincidence circuit of the scintillatio
13. Oo Promega Technical Manual TNT Quick Coupled Transcription Translation Systems All technical literature is available on the Internet at www promega com tbs Please visit the web site to verify that you are using the most current version of this T NT Q u C k C 0 u p l e d Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail techserv promega com Transcription Translation A DESC B nnn 2 Syste m S 2 Product Components and Storage Conditions 0 csecsssseseessseeeeesen 5 3 General Considerations ccccscsssesssesssessesseessessesssecsssesssssecssecsssesecssecsseeseess A DNA Template Considerations B Creating a Ribonuclease Free Environment C Handling Of Lysate cassscsssasssesssscsesssvsssssssssusssnnseivavesensssoussuesienresisinesissnsibeanseid INSTRUCTIONS FOR USE OF PRODUCTS L1170 L1171 L2080 AND L2081 4 Translation Procedure j csssissesssisasscecssssessssessigssnteseisn sssonsecesensassipeocebedisoseie dive 8 A General Protocol for TNT Quick Coupled Transcription Translation Reactions Using Plasmid DNA sssssss 9 B General Protocol for TNT T7 Quick Coupled Transcription Translation Reactions Using PCR Generated DNA 5 Positive Control Translation Reactions Using Luciferase 0 13 A Radioactive Luciferase Control Reaction B Non Radioactive Luciferase Control Reaction
14. System 30 reactions V8830 MagneGST Protein Purification System 40 reactions V8600 200 reactions V8603 HisLink Protein Purification Resin 50ml V8821 HisLink 96 Protein Purification System 1x96 V3680 5 x 96 V3681 Protein Protein Interactions Product Size Cat MagneGST Pull Down System 80 reactions V8870 Plasmid Purification Product Size Cat PureYield Plasmid Midiprep System 25 preps A2492 100 preps A2495 WU S Pat Nos 5 324 637 and 5 492 817 Australian Pat No 660329 and Japanese Pat No 2904583 U S Pat Nos 5 283 179 5 641 641 5 650 289 and 5 814 471 Australian Pat No 649289 European Pat No 0 553 234 and Japanese Pat No 3171595 OU S Pat No 5 552 302 European Pat No 0 422 217 Australian Pat No 646803 and Japanese Pat Nos 3009458 and 3366596 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 fields is needed for any commercial sale of nucleic acid contained within or derived from this product For Laboratory Use Any use of the product for diagnostics requiring clearance or approval by the FDA may require a license under Mayo Clinic U S Pat No 6 027 913 1996 2009 Promega Corporation All Rights Reserved Flexi RNasin TNT and Wizard are registered trademarks of P
15. ansfer 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 define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes Cell 44 283 92 11 Appendix 11 A Composition of Buffers and Solutions acrylamide solution 30 37 5 1 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 dithiothreitol 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 100m1 10 SDS Add deionized water to a final 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 volume of 100ml Store at room temperature stacking gel 4X buffer 6 06g Tris base 4ml 10 SDS Bring the volume
16. ased protein synthesis Incubate the reaction at 30 C the optimal temperature Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 21 Oo Promega O Promega 9 Troubleshooting continued Symptoms Symptoms Causes and Comments Causes and Comments Smearing on the gel Gel not clean Gel must be washed before plac ing onto film Once gel electrophoresis is Unexpected bands present Denaturing temperature too high Denature at higher molecular weights sample at a lower temperature e g 60 80 C or bands stuck in stacking gel for 10 15 minutes Unexpected bands Proteolysis of translation product Add protease present on the gel inhibitors such as 0 2 macroglobulin leupeptin or chymostatin 0 5 1pg 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 Mg or K concentration can increase fidelity 26 The S methionine used is not translational grade or beyond its expiration date There are reports of a 42kDa band with some grades of S methionine 16 We recommend Perkin Elmer EasyTag L S methionine Perkin Elmer Cat NEG709A to avoid this 42kDa band Globin may appear on the autoradiogra
17. aster Mix and incubated in a 50pl 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 gt www promega com Part TM045 Printed in USA Page 2 Revised 3 09 TnT Coupled Reticulocyte TNT Quick Coupled Lysate System Transcription Translation System TNT Rabbit TT Reticulocyte Lysate 4 Add TNT ff Reaction Buffer TNT Quick Master Mix Add TNT T 7 RNA Polymerase 4 Add Amino Acid Mixture Minus Methionine 4 Add RNasin lag Ribonuclease Inhibitor L Add label of choice 4 Add DNA template and Nuclease Free Water 4 Incubate at 30 C for 60 90 minutes 4 Separate translation products by SDS PAGE Detect 1537MB11_2A Figure 1 Comparison of the TNT Coupled Reticulocyte Lys
18. asurement 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 9 Troubleshooting 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 Symptoms Causes and Comments The control reaction Loss of reaction component s activity The produces no luciferase 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 efficiency Certain gene constructs may require different Mg and K concentrations for optimal expression Add 1 3yl 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 decre
19. ate 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 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 3 Oo Promega Oo Promega T 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 Drug screening affecting translation rates Mutation and detection analysis In vitro expression cloning IVEC functional genomics Protein structure analysis Electrophoretic mobility shift assays EMSAs for DNA protein interactions i e enzyme kinetics DNA footprinting and protein e Protein protein interactions using cross linking studies GST pull downs Protein RNA binding assays Immunoprecipitation of protein Post translational modification complexes tests e Protein dimerization assays Verification characterization of e Ligand binding region cloned genes determination confirmation competition assays The TNT Quick Coupled Transcription Translation Systems are also useful for detecting protein protein interactions in vitro
20. ation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 11 Oo Promega Oo Promega 4 C Notes continued 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 Mg 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 1 211 of the T7 TNT PCR Enhancer 4 We recommend using a grade of S methionine PerkinElmer EasyTag L 5S 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 40uCi 1 41 of S 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 10pCi level 1p is sufficient for adequate detection 5 Except for the actual transcription translation incubation all handling of the TNT Qui
21. brand Vitamin e separating gel 4X buffer Assay Grade e stacking gel 4X buffer 5 TCA SDS sample buffer e Whatman GF A e SDS polyacrylamide gels glass fiber filter optional precast Whatman Cat 1820 021 polyacrylamide gels e acetone 7 A Determination of Percent Incorporation of Radioactive Label 1 After the 50pl translation reaction is complete remove 21 from the reaction and add it to 98 of IM NaOH 2 H O 2 Vortex briefly and incubate at 37 C for 10 minutes 3 At the end of the incubation add 900l of ice cold 25 TCA 2 casamino acids to precipitate the translation product Incubate on ice for 30 minutes Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 15 7 A Determination of Percent Incorporation of Radioactive Label continued 4 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 250p1 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 5 For determination of 3S incorporation put the filter in the appropriate scintillation cocktail invert to mix and count in a liquid scintillation counter 6 To d
22. ck 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 lpg of plasmid DNA template is used We recommend using 0 2 2 0pg of plasmid DNA in TNT Quick reactions The use of more than 1pg 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 Transcend tRNA and FluoroTect Green tRNA can be increased 1 411 to allow more sensitive detection of proteins that contain few lysines or are poorly expressed 5 Positive Control Translation Reactions Using Luciferase The assay for firefly 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 modification for enzymatic activity The Luciferase Assay System is a substantial improvement over conventional methods
23. d Solutions B Luciferase SP6 T7 Control DNAs C Related Products 1 Description The TNT Quick Coupled Transcription Translation Systems are convenient single tube coupled transcription translation reactions for eukaryotic in vitro translation The original TNT Coupled Reticulocyte Lysate Systems simplified 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 simplifies 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 significantly 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 configurations for transcription and translation of genes cloned downstream from either the T7 or SP6 RNA polymerase promoters To use these systems 0 2 2 0pg 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 M
24. d 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 Green 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 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 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 Standard Standard Standard Reaction Using Reaction Using Reaction Using Transcend FluoroTect Components S methionine tRNA Green tRNA TNT T7 Quick Master Mix see Note 3 Section 4 C 40pl 40pl 40pl Methionine 1mM mix gently prior to use 1l 1pl S methionine 1 000Ci mmol at 10mCi ml see Note 4 Section 4 C 2pl PCR generated DNA template s see Note 1 Section 4 C 2 5 5pl 2 5 5p
25. d 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 27 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 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 RN
26. eactions 13251 Rabbit Reticulocyte Lysate Wheat Germ Extract Combination System Product Size Cat Rabbit Reticulocyte Wheat Germ Extract Combination System 12 reactions each L4330 E coli 30 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 11130 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 29 Oo Promega 11 C Related Products continued Transcend Non Radioactive Translation Detection Systems Product Size Cat Transcend Colorimetric Non Radioactive Translation Detection System 30 reactions 15070 Transcend Chemiluminescent Non Radioactive Translation Detection System 30 reactions 15080 Transcend Biotinylated tRNA 30pl 15061 For Laboratory Use FluoroTect Green in vitro Translation Labeling System Product Size Cat FluoroTect Green in vitro Translation Labeling System 40 reactions L5001 For Laboratory Use Canine Pancreatic Microsomal Membranes Product Size Cat Canine Pancreatic Microsomal Membranes 50pl Y4041 Protein Purification Product Size Cat MagZ Protein Purification
27. eactions 15020 TNT T7 Quick for PCR DNA 40 reactions 15540 TNT SP6 Coupled Reticulocyte Lysate System Trial Size 8 reactions 14601 TNT T7 Coupled Reticulocyte Lysate System Trial Size 8 reactions 14611 For Laboratory Use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 gt www promega com Part TM045 Printed in USA Page 28 Revised 3 09 TNT Coupled Wheat Germ Extract Systems Product Size Cat 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 For Laboratory Use Rabbit Reticulocyte Lysate Product Size Cat Rabbit Reticulocyte Lysate Nuclease Treated 5 x 200pl 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 5 x 200pl 14540 Bulk Flexi Rabbit Reticulocyte Lysate is available from Promega Wheat Germ Extract Product Size Cat Wheat Germ Extract 5 x 200pl L4380 Wheat Germ Extract Plus 40 x 50pl reactions 13250 10 x 50l r
28. ed 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 Green sin 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 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 5m microcentrifuge tube After adding 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 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 Example of a TNT Quick Reaction Using Plasmid DNA Standard Standard Standard Reaction Using Reaction Using Reaction Using Transcend FluoroTect Components S methionine tRNA Greeny tRNA TNT Quick Master Mix see Note 3 Section 4 C 40pl 40pl 40pl Methionine 1mM mix gently prior to use pl ipl 35S methionine 1 000Ci mmol at 10mCi ml see Note 4 Section 4 C 2ul plasmid DNA template s
29. els Notes 1 Fixing polyacrylamide gels does not interfere with the detection of FluoroTect Green 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 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 Invitrogen Cat A 5770 for immunoprecipitation and Western blot analysis of translation products 7 D 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 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 Radionctive Translation Detection Systems Technical Bulletin 1B182 1 Once the 50pl translation reaction is complete or at any desired time point remove
30. ely The gel 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 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 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 buffer The blot may then be subjected to immunodetection analysis For more information refer to the Promega Protocols and Applications Guide Online Edition 25 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 17 7 C Denaturing Gel Analysis of Translation Products Labeled with the FluoroTect Green in vitro Translation Labeling System The fluorescent translation product should be resolved by SDS PAGE and then visualized
31. embrane 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 11 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 autoradiography or fluorography Fluorography dramatically increases the sensitivity of detection of 3S 14C and 5H 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 Commercial reagents such as Amplify Reagent GE Healthcare Bio sciences 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 speed and the ability to quantitate the radioactive bands 6 Dry the gel before exposure to film as follows Soak the gel in 10 glycerol for 5 minutes to prevent the gel from cracking during drying Place the gel ona 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 complet
32. enience and safety precast gels provide consistent results 1 Once the 50yl translation reaction is complete or at any desired timepoint remove a 1 5pl aliquot and add it to 20 1 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 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 Oo Promega Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Printed in USA Page 16 Revised 3 09 3 A small aliquot 5 10pl of the denatured sample can then be loaded onto an SDS polyacrylamide gel or stored at 20 C It is not necessary to separate abeled 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 m
33. etermine total counts present in the reaction spot a 5pl 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 7 To determine background counts remove 21 from a 50pl 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 filter Step 5 cpm of unwashed filter Step 6 50 x 100 percent incorporation 9 Perform the following calculation to determine the fold stimulation over background cpm of washed filter Step 5 cpm of no DNA control reaction filter Step 7 gt sole Simdata 7 B 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 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 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 conv
34. g l 100p1 T7 TNT PCR Enhancer L1170 only 50pl Methionine 1mM 250p1 Luciferase Assay Reagent e 125ml Nuclease Free Water Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Printed in USA Page 4 Revised 3 09 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 5 Oo Promega Oo Promega 2 Product Components and Storage Conditions continued Product Cat TNT T7 Quick Coupled Transcription Translation System Trial Size L1171 TNT SP6 Quick Coupled Transcription Translation System Trial Size 12081 For Laboratory Use Each system contains sufficient reagents to perform approximately 5 x 50p translation reactions Includes 200p1 TNT Quick Master Mix s 5pg SP6 or T7 Luciferase Control DNA 0 5p l 100p1 T7 TNT PCR Enhancer L1171 only 7 50pl Methionine 1mM Storage and Stability Store all components at 70 C Product components are sensitive to CO avoid prolonged exposure frequent temperature fluctuations and multiple freeze thaw cycles which can adversely affect stability activity and performance Luciferase Assay Reagent LAR is stable for at least 12 months if stored and handled properl
35. hem 275 19422 7 10 FluoroTect Green in vitro Translation Labeling System Technical Bulletin TB285 Promega Corporation Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Printed in USA Page 22 Revised 3 09 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 23 Oo Promega 10 References continued 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 Identification of two critical base pairings in 5 untranslated regions affecting translation efficiency of synthetic uncapped globin mRNAs Biochim Biophys Acta 1130 29 37 Q Jackson R J and Standart N 1990 Do the poly A tail and 3 untranslated region control mRNA translation Cell 62 15 24 e Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY ao 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 a Jackson RJ and Hunt T
36. 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 S methionine TNT Quick Master Mix see Note 3 Section 4 C 40ul S methionine 1 000Ci mmol at 10mCi ml see Note 4 Section 4 C ul Appropriate Luciferase Control DNA 0 5pg pl see Section 11 B 2ul Nuclease Free Water to a final volume of 50pl 2 Incubate the reaction at 30 C for 60 90 minutes see Note 3 Section 4 C 3 Analyze the results of translation by measuring direct incorporation of radiolabel Section 7 A and or gel analysis of translation products Section 7 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 5 B Non Radioactive Luciferase Control Reaction 1 The following example contains Methionine TNT Quick Master Mix see Note 3 Section 4 C 40l Methionine 1mM pl Appropriate Luciferase Control DNA 0 5pg pl see Section 11 B ul Nuclease Free Water to a final volume of 5Opl 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
37. iption Translation System For most constructs optimal results are obtained when 11g of plasmid DNA template is used However we have used 0 2 2 0ug of DNA template and obtained satisfactory levels of translation The use of more than 1pg 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 Sacll BstXI Nesil Apal and Aatll 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 11 of the T7 TNT PCR Enhancer in each 50pl 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 first 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 gt www promega com Part TM045 Printed in USA Page
38. l 2 5 5pl T7 TNT PCR Enhancer see Note 2 Section 4 C Tpl Tyl 1pl Transcend Biotin Lysyl tRNA see Note 9 Section 4 C 1 2pl FluoroTect Greeny tRNA see Note 9 Section 4 C 1 2pl Nuclease Free Water to a final volume of 50pl 50pl 50pl 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 Procedures for determination of radiolabel incorporation Section 7 A and SDS PAGE analysis of translation products Section 7 B are provided If using FluoroTect Green tRNA see Section 7 C for Transcend tRNA reactions see Section 7 D 4 C Notes 1 PCR generated templates can be used directly from the amplification reaction We recommend using 2 5 5pl from the amplification reaction but up to 7pl can be used in a 50pl 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 11 of the T7 TNT PCR Enhancer in a 50pl reaction to increase transcription translation when using PCR generated DNA linear plasmid or viral enhanced plasmids Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 gt www promega com Part TM045 Printed in USA Page 10 Revised 3 09 Promega Corpor
39. loss of luciferase activity Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 gt www promega com Part TM045 Printed in USA Page 12 Revised 3 09 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 13 Oo Promega 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 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 e Canine Pancreatic Microsomal Membranes Cat Y4041 S methionine 1 000Ci mmol at 10mCi ml 1 Remove the reagents from the freezer and allow them to thaw on ice Note
40. m or stained gel It appears as a broad band migrating at 10 15kDa Aminoacyl tRNAs may produce background bands 25kDa Add RNase A to the lysate reaction after completion to a final concentration of 0 2mg ml Incubate for 5 minutes at 30 C Oxidized B mercaptoethanol is present or not enough SDS in the loading buffer Use a loading buffer that contains 2 SDS and 100mM DTT Unexpected bands present when A nickel based resin is used to purify isolating polyhistidine tagged protein polyhistidine tagged proteins Hemoglobin present in the rabbit reticulocyte lysate will bind to the nickel and co elute with the polyhistidine tagged protein Use the MagZ Protein Purification System Cat V8830 or an alternate purification tag to isolate the protein from the TNT lysate and avoid this problem 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 samples loaded on the gel and the amount of loading buffer 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 10 References 1 Pelham H R B and Jackson R J 1976 An efficient mRNA dependent translation system from reticulocyte lysates
41. mega offers TNT T7 Quick for PCR DNA Cat L5540 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 efficiency 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 fivefold 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 SV Minipreps DNA Purification System or the PureYield Plasmid Midiprep System DNA prepared by the standard alkaline lysis method described by Sambrook Fritsch and Maniatis 14 is also sufficiently clean for use in the TNT Quick Coupled Transcr
42. n 1 WwW EM W EM W Wash E Eluate M Marker z 2598MA03_9A Autoradiography Western L J Figure 2 The study of protein protein interactions using the TNT Systems 6 This schematic shows translation of one protein with radioactive 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 proteins and is often used to verify the in vivo results obtained from yeast two hybrid experiments Promega offers the MagneGST Pull Down System Cat V8870 for GST pull down experiments 2 Product Components and Storage Conditions Product Cat TNT T7 Quick Coupled Transcription Translation System L1170 TNT SP6 Quick Coupled Transcription Translation System 12080 For Laboratory Use Each system contains sufficient reagents to perform approximately 40 x 501 translation reactions Includes 1 6ml TNT Quick Master Mix 8 x 200p 5pg SP6 or T7 Luciferase Control DNA 0 5p1
43. n 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 S methionine radioactive Transcend Non Radioactive Detection System colorimetric or chemiluminescent or FluoroTect Green Systems fluorescent 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 e 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 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 Note Technical Manuals and Bulletins are available online at www promega cony tbs or by request from Technical Services 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 e Radiolabel
44. n 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 cpm minus background cpm ie sample background 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 A The sample may be placed directly in the scintillation vial if it Oo Promega Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Printed in USA Page 20 Revised 3 09 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 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 me
45. ns will be suitable for most applications the efficiency 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 8p1 of Canine Microsomal Membranes Transcription Translation may be inhibited by as much as 50 with 0 6p1 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 reactions alone Depending on the construct used protein synthesis efficiency can be expected to drop between 10 50 in the presence of Microsomal Membranes 5 In some cases it is difficult to determine if efficient 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 e IM NaOH 2 H O e Whatman 3MM filter paper e 25 TCA 2 casamino e 30 acrylamide solution acids Difco
46. onal description Amp B 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 so 1767 1796 pUC M13 reverse primer 17mer 1833 1817 pUC M13 reverse primer 22mer 1838 1817 B lactamase gene Amp 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 Phone 608 274 4330 Fax 608 277 2516 gt www promega com Part TM045 Printed in USA Page 26 Revised 3 09 Sacl 1767 dA dT 35 luc Luciferase T7 Xmnl Control DNA 2632 4331bp Amp BamHI 44 Notl 22 Hindlll 11 T7 Initiation 1 d ori T7 Promoter 1916VA04_6A 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 il GLPrimer2 52 74 Luciferase gene 51 1700 Poly A dA 1770 1799 B lactamase gene Amp 2444 3304 T7 RNA polymerase promoter 4315 3 T7 RNA polymerase promoter primer 4315 3 11 C Related Products The in vitro synthesis of proteins is a popular method in biological research Among other applications translation systems are used to rapidly characterize plasmi
47. rane 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 buffer Instructions for chemiluminescent detection of products are found in Section 5 D of the Transcend Non Radioactive Translation Detection Systems Technical Bulletin TB182 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM045 Printed in USA Page 18 Revised 3 09 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 19 Oo Promega 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
48. romega Corporation FluoroTect MagneGST MagZ PureYield and Transcend are trademarks of Promega Corporation Amplify Fluorlmager 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 FMBIO is a registered trademark of Hitachi Software Engineering Company Ltd Immobilon is a trademark of Millipore Corporation Novex is a registered trademark of Invitrogen Corporation X OMAT is a registered trademark of Eastman Kodak Co Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 gt www promega com Part TM045 Printed in USA Page 30 Revised 3 09 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM045 Revised 3 09 Page 31
49. y 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 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 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 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 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 efficiencies Note For coupled transcription translation from PCR generated templates Pro
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