Protocols and Applications Guide (US Letter Size) - Promega

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||||| 5Protein Expression Table 5.2. Summary of Coupled Transcription/Translation Systems. Template Expected Yield1 150–300ng Kozak Preferred X RBS Required – PCR-Generated Not Recommended Circular DNA X Linear DNA X2 System TNT® Coupled Reticulocyte Lysate Transcription/Translation (T7, T3 or SP6 RNA Polymerase) (Cat.# L4610, L4950, L4600) 200–350ng X – X 3 X X 3 TNT® Quick Coupled Transcription/Translation (T7/SP6 RNA Polymerase) (Cat.# L1170, L2080) 150–300ng X – X Not Recommended X TNT® T7 Quick for PCR DNA (Cat.# L5540) 1–4µg X – Not Recommended X Not Recommended TNT® Insect Cell Extract Protein Expression System (Cat.# L1101) 1–5µg Labeling Options Met, Cys, Leu, FluoroTect, Transcend Met, FluoroTect, Transcend Met, FluoroTect, Transcend Met, Cys, Leu, Transcend, FluoroTect Met, Cys, Leu, Transcend, FluoroTect Met, Cys, Leu, FluoroTect Met, Cys, Leu, Transcend, FluoroTect Met, Cys, Leu, Transcend, FluoroTect Met, Cys, Leu, Transcend, FluoroTect Met, Cys, Leu, Transcend, FluoroTect Protocols & Applications Guide www.promega.com rev. 6/09 X – X X X TNT®SP6 High-Yield Wheat Germ Protein Expression System (Cat.# L3260) 150–300ng X – X X 5 X 4 TNT® Coupled Wheat Germ (T7, T3 and SP6 RNA Polymerase)(Cat.# L4120, L4130, L4140) ~300ng – X Not Recommended X Not Recommended E. coli T7 S30 Extract for Circular DNA (Cat.# L1130) 150–200ng – X Not Recommended X Not Recommended E. coli S30 for Circular DNA (Cat.# L1020) 100–250ng – X X Not Recommended E. coli S30 for Linear DNA (Cat.# L1030) 10–25µg – X Not Recommended X Not Recommended S30 T7 High-Yield Protein Expression System (Cat.# L1110) 1 Expected yield for 50µl reaction volumes. 2 Only T7 linear templates. 3 T7 promoter only, must use provided T7 TNT® PCR Enhancer. 4 Must be linearized for T7. PROTOCOLS & APPLICATIONS GUIDE 5-4
||||| 5Protein Expression Coupled Systems, since the master mix contains all amino acids except methionine, and the labeling efficiency with other amino acids will be significantly reduced. Table 5.3. Recommended Concentrations of Alternative Radiolabeled Amino Acids. Final Concentration Volume Amino Acid in Reaction Required [3H]leucine (100–200Ci/mmol) 0.5mCi/ml 5µl [14C]leucine (300mCi/mmol) [35S]cysteine (1,200Ci/mmol) 5µCi/ml 0.3mCi/ml 5µl 5µl [35S] Methionine We recommend using a “translational grade” [35S]methionine such as PerkinElmer EasyTag L-[35S]methionine (PerkinElmer Cat.# NEG709A). We have obtained acceptable results using 1–4µl of [35S]methionine (1,200Ci/mmol at 10mCi/ml). Depending upon the translational efficiency of the experimental RNA/DNA and number of methionines present in the protein, the amount of [35S]methionine can be adjusted to balance exposure time against label cost. When using and storing [35S]methionine, follow the manufacturer’s recommendations. Non-Radioactive Protein Labeling The Transcend Non-Radioactive Translation Detection Systems (Cat.# L5070 and L5080) and the FluoroTect GreenLys in vitro Translation Labeling System (Cat.# L5001) can be used with any of the TNT® Coupled or Quick Coupled Systems. These systems use a precharged lysine tRNA, which is incorporated into the translated protein. The Transcend System incorporates a biotinylated lysine, which can be detected by blotting and probing with streptavidin/AP or streptavidin HRP. The FluoroTect Reagent incorporates a fluorescently labeled lysine (BODIPY®), which can be detected directly in the gel. III. Eukaryotic Cell-Free Expression Systems The TNT® Coupled Reticulocyte Lysate Systems offer researchers an alternative for eukaryotic in vitro transcription and translation: a one-tube, coupled transcription/translation system. Standard Rabbit Reticulocyte Lysate or Wheat Germ Extract translations (Pelham and Jackson, 1976) commonly use RNA synthesized in vitro (Krieg and Melton, 1984) from SP6, T3 or T7 RNA polymerase promoters. The RNA is then used as a template for translation. The TNT® Systems bypass many of these steps by incorporating transcription directly in the translation mix. In most cases, the TNT® System reactions produce significantly more protein (two- to sixfold) in a 1- to 2-hour reaction than standard in vitro Rabbit Reticulocyte Lysate Protocols & Applications Guide www.promega.com rev. 6/09 or Wheat Germ Extract translations using RNA templates. In addition, TNT® Lysates also can be used with microsomal membranes to study processing events (see Section VII). A. TNT® SP6 High-Yield Protein Expression System The TNT® SP6 High-Yield Protein Expression System uses a high-activity wheat germ extract supplemented with SP6 RNA polymerase and other components to express 10–100µg/ml of soluble protein in a one-tube coupled transcription/translation format. The system produces substantially more protein than conventional wheat germ systems while maintaining the ease of use that cell-free and coupled transcription and translation conditions afford. Following a two-hour incubation, the expressed protein can be used directly in downstream applications; no protein purification steps are necessary. For a detailed protocol and background information on this system, please see Technical Manual #TM282 (www.promega.com /tbs/tm282/tm282.html). B. TNT® T7 Insect Cell Extract Protein Expression System The TNT® T7 Insect Cell Extract Protein Expression System is a single-tube, coupled transcription/translation system for the cell-free expression of proteins. The extract is made from the commonly used Spodoptera frugiperda Sf21 cell line (Ezure et al. 2006) However, the extract contains no endogenous glycosylation machinery. All necessary components are present in the TNT® T7 ICE Master Mix (ICE = insect cell extract). Protein is expressed from genes cloned downstream of the T7 polymerase promoter. The best expression will be achieved using vectors that contain the baculovirus polyhedrin 5´ and 3´ untranslated region (Suzuki et al. 2006). Two vectors, the pF25A and pF25K ICE T7 Flexi® Vectors (Cat.# L1061 and L1081), have been designed specifically to produce optimal protein yields in this system. For a detailed protocol and background information on this system, please see Technical Manual #TM305 (www.promega.com/tbs/tm305/tm305.html). The pF25A and pF25K ICE T7 Flexi® Vectors are compatible with the Flexi® Cloning System (Cat.# C8640), which allows easy transfer of sequences to and from additional Flexi® Vectors. Additional Flexi® Vectors are available for use with other protein expression systems. For a detailed protocol and background information on this system, please see Technical Manual #TM254 (www.promega.com /tbs/tm254/tm254.html). C. TNT® Coupled Wheat Germ Extract Systems—Coupled Transcription/Translation The TNT® Wheat Germ Extract Systems are available in five configurations for transcription and translation of genes cloned downstream from the SP6, T3 or T7 RNA polymerase promoter. With these systems, a 50µl reaction is programmed with 0.2–2µg of template and incubated for 1.5 hours at 30°C. For a detailed protocol and background information about this system, please see Technical Bulletin #TB165 (www.promega.com /tbs/tb165/tb165.html). PROTOCOLS & APPLICATIONS GUIDE 5-5
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CONTENTS I. Nucleic Acid Amplificat
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| 1Nucleic Acid Amplification Unamp
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| 1Nucleic Acid Amplification inclu
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| 1Nucleic Acid Amplification 3. Pl
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| 1Nucleic Acid Amplification 13. S
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| 1Nucleic Acid Amplification IX. R
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| 1Nucleic Acid Amplification Hoppe
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|| 2RNA Interference CONTENTS I. RN
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|| 2RNA Interference zebrafish (War
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|| 2RNA Interference Target Site Se
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|| 2RNA Interference Transient Tran
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|| 2RNA Interference VII. Reference
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|| 2RNA Interference Wianny, F. and
Page 47 and 48: ||| 3Apoptosis I. Introduction A. C
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Page 133 and 134: ||||||| 7Cell Signaling CONTENTS I.
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|||||||| 8Bioluminescence Reporters
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Table 8.1. pGL4 Luciferase Reporter
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||||||||| 9DNA Purification I. Intr
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||||||||||||| 13Cloning CONTENTS I.
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