9 gen 2022 — translation extract (e.g., Rabbit Reticulocyte Lysate, E. coli S30 ... Galacto-Light™ β-Galactosidase Reporter Assay System Instruction Manual, Tropix, Inc.
TECHNICAL BULLETIN TranscendTM Non-Radioactive Translation Detection System Instructions for Use of Products L5061 and L5070 Revised 8/21 TB182 TranscendTM Non-Radioactive Translation Detection System 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 Bulletin. E-mail Promega Technical Services if you have questions on use of this system: techserv@promega.com 1. Description.......................................................................................................................................... 2 2. General Considerations........................................................................................................................ 4 2.A. Effect of Biotinylated Lysine Incorporation on Expression Levels and Enzyme Activity...................... 4 2.B. Estimating Incorporation Levels of Biotinylated Lysine in TranscendTM Reactions............................ 4 3. Product Components and Storage Conditions......................................................................................... 6 4. Biotinylated Lysine Incorporation Using TranscendTM tRNA.................................................................... 6 4.A. Translation Protocol.................................................................................................................... 6 4.B. In Vitro Translation Systems........................................................................................................ 8 5. Post-Translational Analysis................................................................................................................... 9 5.A. Denaturing Gel Analysis of Translation Products............................................................................ 9 5.B. Electroblotting of Proteins to Membrane..................................................................................... 10 5.C. Colorimetric (BCIP/NBT) Detection........................................................................................... 10 6. Troubleshooting................................................................................................................................ 11 7. References......................................................................................................................................... 12 8. Appendix........................................................................................................................................... 13 8.A. Composition of Buffers and Solutions.......................................................................................... 13 8.B. Related Products....................................................................................................................... 13 8.C. Summary of Changes................................................................................................................. 14 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 1 www.promega.com TB182 · Revised 8/21 1. Description The TranscendTM Non-Radioactive Translation Detection Systems allow nonradioactive detection of proteins synthesized in vitro. Using these systems, biotinylated lysine residues are incorporated into nascent proteins during translation, eliminating the need for labeling with [35S]methionine or other radioactive amino acids. This biotinylated lysine is added to the translation reaction as a precharged, -labeled biotinylated lysine-tRNA complex (TranscendTM tRNA) rather than a free amino acid (Figure 1). After SDS-PAGE and electroblotting, the biotinylated proteins can be visualized by binding either Streptavidin-Alkaline Phosphatase (Streptavidin-AP) or Streptavidin-Horseradish Peroxidase (Streptavidin-HRP), followed by colorimetric detection. Typically, 0.55ng of protein can be detected by these methods within 34 hours after gel electrophoresis (Figure 2). The use of TranscendTM tRNA offers several advantages: · No radioisotope handling, storage or disposal is needed. · The biotin tag is sensitive (0.55ng). · The biotin tag is stable for 12 months, both as the TranscendTM tRNA reagent and within the labeled proteins. It is not necessary to periodically resynthesize biotin-labeled proteins, unlike 35S-labeled proteins whose label decays over time. · Results can be visualized quickly using colorimetric detection. The precharged E. coli lysine tRNAs provided in this system have been chemically biotinylated at the -amino group using a modification of the methodology developed by Johnson et al. (1). The biotin moiety is linked to lysine by a spacer arm, which greatly facilitates detection by avidin/streptavidin reagents (Figure 1). The resulting biotinylated lysine tRNA molecule (TranscendTM tRNA) can be used in either eukaryotic or prokaryotic in vitro translation systems such as the TNT® Coupled Transcription/Translation Systems, Rabbit Reticulocyte Lysate System or E. coli S30 Extract System (2,3). Lysine is one of the more frequently used amino acids. On average, lysine represents 6.6% of a proteins amino acid content, whereas methionine represents only 1.7% (4). lysine spacer arm biotin A OCO NH C C NH3 S NO C N O tRNA 0877MA10_3A Figure 1. Structure of TranscendTM tRNA. 2 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 TB182 · Revised 8/21 www.promega.com Standard radioisotopic incorporation and detection M* M* Translation with incorporation of 35S-met (1 hour) SDS PAGE (1 hour) TranscendTM Biotinylated Lysine tRNA incorporation and detection LL L Translation with incorporation of biotinylated lysine (1 hour) SDS PAGE (1 hour) Fix gel (30 minutes) Transfer to PVDF or nitrocellulose membrane (1 hour) Treat with enhancer (30 minutes) Block, bind Strep-AP, wash (2 hours) Dry gel (1 hour) Expose to X-ray film (410 hours) Add Western Blue® Substrate to develop colored bands (110 minutes) Colorimetric detection 0878MA08_3A Time required = 814 hours Time required = 6 hours Figure 2. Comparison of incorporation and detection protocols using radiolabeled amino acids or TranscendTM tRNA. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 3 www.promega.com TB182 · Revised 8/21 2. General Considerations 2.A. Effect of Biotinylated Lysine Incorporation on Expression Levels and Enzyme Activity Lysine residues are common in most proteins and usually are exposed at the aqueous-facing exterior. The presence of biotinylated lysines may or may not affect the function of the modified protein. For example, the enzymatic activity of -galactosidase is reduced by the incorporation of biotinylated lysines, but luciferase and chloramphenicol acetyltransferase (CAT) are less affected (Figure 3 and Table 1). In gel shift experiments, c-Jun synthesized in TNT® Coupled Reticulocyte Lysate reactions and labeled with TranscendTM tRNA performed identically to unlabeled c-Jun (3). 2.B. Estimating Incorporation Levels of Biotinylated Lysine in TranscendTM Reactions The incorporation of radioactively labeled amino acids into proteins is typically quantitated as a percent incorporation of the label added. This value can include incorporation of radioactivity into spurious gene products such as truncated polypeptides. Thus, percent incorporation values provide only a rough estimate of the amount of full-length protein synthesized and do not provide any information on translation fidelity. With TranscendTM reactions, it is difficult to determine the percent incorporation of biotinyl-lysines into a translated protein directly. An alternative means of estimating translation efficiency and fidelity in TranscendTM reactions is to determine the minimum amount of product detectable after SDS-polyacrylamide gel electrophoresis. We have been able to detect proteins in 1l of translation reaction using as little as 0.5l of TranscendTM tRNA per 50l reaction (Figure 3). The amount of biotin incorporated into translation products increases linearly with the amount of TranscendTM tRNA added to the reaction, up to a maximum of about 2l. -galactosidase luciferase µg TranscendTM tRNA 1.0 0.5 0.25 0 1.0 0.5 0.25 0 chloramphenicol acetyltransferase 1.0 0.5 0.25 0 colorimetric 0865TA Figure 3. Effects of TranscendTM tRNA concentration on detection of proteins synthesized in vitro. TNT® Coupled Transcription/Translation reactions were performed according to the directions (5). The indicated amounts of TranscendTM tRNA (equivalent to 2.0, 1.0, 0.5 or 0l) were added to the translation reactions prior to incubation at 30°C for 1 hour. One microliter of the reaction was used for SDS-PAGE. The separated proteins were transferred to PVDF membrane (100V for 1 hour). The membrane was blocked in TBS + 0.5% Tween® 20 (TBST) for 1 hour, probed with Streptavidin-AP (45 minutes), washed twice with TBST and twice with water, and incubated with Western Blue® Substrate for 2 minutes. 4 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 TB182 · Revised 8/21 www.promega.com Although we have not determined the ratio of moles of biotin incorporated per mole of protein, experiments by Crowley et al. using fluorescently labeled tRNA (-NBD-[14C]lysine tRNA) indicate that 2533% of lysine residues are biotinylated in translation products that contain 46 lysine residues (6). Although proteins vary in lysine composition and each molecule synthesized in the translation reaction will contain a different number of biotinylated lysines, it is reasonable to expect that similar levels of labeling (2035% of lysines) will be obtained with other proteins. Table 1. Effect of Biotinylated Lysine Incorporation on Levels of Synthesis and Enzymatic Activity of Three Reporter Genes Expressed in the TNT® Reticulocyte Lysate System. TranscendTM tRNA Added to Reaction Level of Synthesis1 (% of Control Reaction) Biotin Signal Strength2 (Colorimetric) Enzymatic Activity3 (% of Control Reaction) Luciferase (40 total lysines) 0.5l 101 44 101 1.0l 93 136 93 2.0l 78 158 94 CAT (12 total lysines) 0.5l 95 1.0l 92 2.0l 73 13 105 70 136 115 -galactosidase (20 total lysines) 0.5l 82 42 53 1.0l 70 82 61 2.0l 70 184 61 These data, taken from a single experiment, are representative of results routinely obtained in our laboratories. 1 [35S]methionine incorporation as a percent of control translation reaction containing no TranscendTM tRNA. 2 Western Blue® Stabilized Substrate (BCIP/NBT) colorimetric detection of bound Streptavidin-AP expressed in arbitrary intensity units obtained with an AMBISTM Image Analysis System. 3 Expressed as a percent of activity obtained with a control translation reaction containing no TranscendTM tRNA. Luciferase activity was determined according to reference 7. CAT activity was determined according to reference 8. -galactosidase activity was quantitated using a chemiluminescent assay (9). Equal volumes of TNT® translation reaction products were used in all assays. Promega Corporation · 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 TB182 · Revised 8/21 3. Product Components and Storage Conditions PRODUCT C AT. # TranscendTM Colorimetric Non-Radioactive Translation Detection System L5070 Each system contains sufficient reagents to label 30 × 50l translation reactions and perform colorimetric detection of biotinylated proteins on 6 blots (7 × 9cm) using Streptavidin-AP Conjugate and Western Blue® Substrate. Includes: · 30l TranscendTM tRNA · 36l Streptavidin-AP · 35ml Western Blue® Stabilized Substrate for Alkaline Phosphatase PRODUCT SIZE TranscendTM tRNA 30l Thirty microliters of TranscendTM tRNA is sufficient for 30 × 50l translation reactions. C AT. # L5061 Note: TranscendTM tRNA (Cat.# L5061) is shipped separately from the other components due to differences in storage temperatures. Storage Conditions: Store TranscendTM tRNA at less than 65°C. Do not subject TranscendTM tRNA to more than 5 freeze-thaw cycles and quickly return it to less than 65°C after use. Store all other components at +2°C to +10°C. Do not freeze Western Blue® Substrate or Streptavidin-Alkaline Phosphatase. 4. Biotinylated Lysine Incorporation Using TranscendTM tRNA 4.A. Translation Protocol Materials to Be Supplied by the User · RNasin® Ribonuclease Inhibitor (Cat.# N2111) or Recombinant RNasin® Ribonuclease Inhibitor (Cat.# N2511) · Nuclease-Free Water (Cat.# P1193) · translation extract (e.g., Rabbit Reticulocyte Lysate, E. coli S30 Extract, TNT® Coupled Transcription/Translation Systems; see Note 1 at the end of this section) · salts, DTT and other components as needed to optimize translation reaction · complete amino acid mix or a combination of two minus amino acid mixes Use the following protocol as a guideline for setting up a translation reaction using TranscendTM tRNA. In general, TranscendTM tRNA may be used in an in vitro translation protocol at a concentration of 12l TranscendTM tRNA per 50l reaction. An example of a standard reaction for Rabbit Reticulocyte Lysate is provided. For more information on specific in vitro translation systems, please see Section 4.B. 6 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 TB182 · Revised 8/21 www.promega.com 1. Remove the translation and TranscendTM tRNA reagents from storage at less than 65°C. Thaw the TranscendTM tRNA on ice. Thaw the translation lysate by hand warming and immediately place on ice. The other components can be thawed at 37°C and then stored on ice as soon as they thaw. Note: Do not subject TranscendTM tRNA to more than 5 freeze-thaw cycles. If necessary, store TranscendTM tRNA in multiple aliquots at less than 65°C. 2. On ice, set up 50l translation reactions as you would for radioactive amino acid incorporation, with the following exception: Add 1l of a complete amino acid mix (containing 1mM of each amino acid) or a combination of two minus amino acid mixtures (such as 0.5l of minus methionine and 0.5l of minus leucine). Note: In the E. coli S30 Extract Systems, the use of a minus lysine amino acid mixture may significantly reduce the yield of translation product. Example of a Standard Rabbit Reticulocyte Lysate Reaction Using TranscendTM tRNA. Rabbit Reticulocyte Lysate 35l Nuclease-Free Water 10l RNasin® Ribonuclease Inhibitor (40u/l) 1l 1mM complete amino acid mixture (or mixture of two minus amino acid mixtures) 1l RNA template in Nuclease-Free Water (see Note 3 at the end of this section) 2l TranscendTM tRNA (see Note 2 at the end of this section) 12l final volume 50l 3. Add all components except the TranscendTM tRNA and gently mix by pipetting the reaction while stirring the reaction with the pipette tip. If necessary, spin briefly in a microcentrifuge to return the sample to the bottom of the tube. Add the TranscendTM tRNA. Note: We recommend including a control reaction containing TranscendTM tRNA but no added nucleic acid template. This allows measurement of any background incorporation contributed by endogenous mRNA and also reveals any endogenous biotinylated protein(s) in the translation extract (see Note 1 at the end of this section). If using a wheat germ extract, this is especially important. 4. Immediately incubate the translation reaction at 30°C for 60 minutes (see Note 4). 5. Terminate the reaction by placing on ice. If necessary, the translation reaction mix can be stored for several months at 20°C to 70°C. 6. Analyze the results of translation. Procedures for gel analysis of translation products are provided in Section 5. Promega Corporation · 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 TB182 · Revised 8/21 Notes: 1. Commonly used translation extracts contain endogenous biotinylated proteins, which may be detected when translation products are analyzed by SDS-PAGE, electroblotting and Streptavidin-AP/Streptavidin-HRP detection. Rabbit Reticulocyte Lysate contains one biotinylated protein, which migrates as a faint band at 100kDa and, in some lots, an additional very faint band at 47kDa. E. coli S30 Extract contains one endogenous protein, migrating at 22.5kDa. Wheat Germ Extract contains five major endogenous biotinylated proteins, migrating at 200kDa, 80kDa, 32kDa and a doublet at 17kDa. A comparison to a no-template control will distinguish the endogenous biotinylated protein(s) from the newly synthesized biotinylated translation product. 2. Biotin labeling of poorly expressed proteins or proteins containing few lysines can be increased by doubling the amount of TranscendTM tRNA added per 50l translation reaction (Table 1 and Figure 3). 3. For maximal expression of your protein, optimize the amount of template added to the reaction and use highly purified RNA or DNA, depending on the translation system used. Translation: An unfractionated cytoplasmic RNA preparation is 6070% rRNA and, as a result, translates poorly. Usually such preparations yield no better than 2030% of the maximum incorporation attainable, and concentrations of 100200g/ml (final concentration) are needed to stimulate translation. In contrast, viral RNAs and poly(A)+ mRNAs (including mRNA transcribed in vitro) can be used at much lower concentrations (580g/ml final concentration). Increased expression levels may be obtained by optimizing the Mg2+ and K+ concentrations in the translation reaction or by using a TNT® Coupled Transcription/Translation System or a TNT® Quick Coupled Transcription/Translation System. Coupled Transcription/Translation: In E. coli S30 reactions, increased band intensities often can be obtained by using 23 times the DNA concentration normally recommended. 4. The appropriate incubation temperature will vary from one translation system to another. Please refer to the appropriate Promega protocol (see Section 4.B) for specific reaction conditions. 4.B. In Vitro Translation Systems For more information on specific in vitro translation systems, please request the appropriate protocol from Promega. · TNT® Quick Coupled Transcription/Translation System Technical Manual (#TM045) · TNT® Coupled Reticulocyte Lysate Systems Technical Bulletin (#TB126) · TNT® Coupled Wheat Germ Extract Systems Technical Bulletin (#TB165) · Flexi® Rabbit Reticulocyte Lysate System Technical Bulletin (#TB127) · Wheat Germ Extract System Technical Manual (#TM230) · E. coli S30 Extract System for Circular DNA Technical Bulletin (#TB092) · E. coli S30 Extract System for Linear Templates Technical Bulletin (#TB102) · E. coli T7 S30 Extract System for Circular DNA Technical Bulletin (#TB219) · TNT® Quick for PCR DNA Technical Manual (#TM235) The number of lysines in the translated polypeptide and the efficiency of translation are the two most important factors affecting the SDS-PAGE band intensity of the translation product. To increase the intensity of weak bands, increase the amount of TranscendTM tRNA to up to twice the standard amount (see Figure 3). 8 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 TB182 · Revised 8/21 www.promega.com To reduce the chance of RNase contamination, wear gloves throughout the experiment and use microcentrifuge tubes and pipette tips that have been autoclaved and handled only with gloves. Addition of RNasin® Ribonuclease Inhibitor to the translation reaction is recommended but not required. RNasin® Ribonuclease Inhibitor prevents degradation of sample mRNAs by many contaminating RNases. If the amount of translation product must be estimated, add radioactive amino acid(s), in addition to the TranscendTM tRNA, to either a control translation reaction or all translation reactions. The percent incorporation of the radioactive amino acid can be used in combination with knowledge of the protein's amino acid composition to estimate the amount of translation product produced. 5. Post-Translational Analysis 5.A. Denaturing Gel Analysis of Translation Products 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 Section 5.C). 1. Once the 50l translation reaction is complete (or at any desired time point), remove a 1l aliquot and add it to 15l of SDS sample buffer. The remainder of the reaction may be stored at 20°C. 2. Close the tube and heat at 70°C for 15 minutes to denature the proteins. 3. Load the denatured sample on an SDS-polyacrylamide gel. (Protocols for SDS polyacrylamide gel electrophoresis may be found in the technical literature provided with Promega translation systems, which is listed in Section 4.B.) 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 2l 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. When loading more than 1l of an S30 Extract translation reaction, first precipitate the proteins by adding 20l of acetone per 5l of extract and incubate on ice for 15 minutes. Centrifuge the acetone-precipitated S30 reaction at 12,000 × g for 5 minutes. Remove the supernatant and dry the pellet. Resuspend the pellet in 5l of water, add 10l of SDS sample buffer, heat at 90100°C for 5 minutes and load 515l of the sample on the gel. Promega Corporation · 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 TB182 · Revised 8/21 5.B. Electroblotting of Proteins to Membrane For colorimetric detection (see Section 5.C), the translation reaction products can be blotted from the SDS-polyacrylamide gel to (in decreasing order of preference) PVDF, nitrocellulose or another membrane using any standard apparatus and protocol, including semi-dry systems. Detailed procedures for electrophoretic blotting are usually included with commercial devices. We 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 prewetted in methanol before it is equilibrated in transfer buffer. 5.C. Colorimetric (BCIP/NBT) Detection Materials to Be Supplied by the User (Solution compositions are provided in Section 8.A.) · PVDF or nitrocellulose membrane · Tris-buffered saline (TBS) · TBS + 0.5% Tween® 20 (TBST) · optional: Ponceau S stain (Sigma Cat.# P7170) Do not allow the membranes to dry out during any of the subsequent steps. Perform all of the washing and incubation steps at room temperature with gentle shaking. Use a shallow container that is slightly larger than the membrane. For the Streptavidin-AP incubation and the color development reaction, use just enough solution to submerge the membrane, protein side up. Usually, this volume is about 0.24ml/cm2 of membrane surface (15ml for a 7 × 9cm membrane). Use at least twice this volume for blocking and washing steps. Determination of Apparent Molecular Weight of Biotinylated Proteins If you are not using biotinylated protein size standards, you may still detect the positions of protein standards by staining the blot after transfer. Immediately after transfer, incubate the blot for 30 seconds in Ponceau S and then destain the blot for 1 minute with water. Mark the locations of the markers with a pencil. Another approach to determining the molecular weights of biotinylated proteins is to run duplicate sets of samples on the left and right halves of the gel. After transfer to a membrane, analyze one set with Streptavidin-AP and stain the other with Amido Black (for nitrocellulose membranes) or Coomassie® blue (for PVDF membranes). Notes: 1. Perform all steps at room temperature. 2. Wear gloves when handling the membrane. Do not let the membrane dry out between steps. 3. After transferring proteins to PVDF membrane, it can be dried and stored. The PVDF membrane must be rewet with 100% methanol and washed twice with deionized water prior to blocking the membrane. 10 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 TB182 · Revised 8/21 www.promega.com Blocking the Membrane 1. Add 15ml of TBST and incubate at room temperature for 60 minutes. Streptavidin-AP Binding 2. Immediately prior to use, dilute 6l of Streptavidin-AP into 15ml of fresh TBST. Pour off the TBST and add this Streptavidin-AP solution to the blot. Rock or agitate gently for 4560 minutes. 3. Pour off the Streptavidin-AP solution. Wash two times for 1 minute each with 15ml of TBST and then two more times (1 minute each) with 15ml of water. Color Development 4. Start the color reaction by incubating the membrane in Western Blue® Stabilized Substrate for Alkaline Phosphatase (or another BCIP/NBT reagent) until the bands of interest have reached the desired intensity. One 7 × 9cm blot requires about 5ml of substrate solution. Protect the solution from strong light. Reactive areas will turn purple, usually within 115 minutes. 5. When the color has developed to the desired intensity, stop the reaction by washing the membrane in deionized water for several minutes, changing the water at least once. 6. Air-dry the membrane. Protect the membrane from light during prolonged storage. 6. 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 No signal develops Poor transfer of proteins to the membrane. Make sure the protocol steps (Section 5.C) were followed correctly. Use a prestained protein marker to confirm good protein transfer. Alternatively, use a protein stain on unblocked membrane to confirm good protein transfer. Poor translation reaction: · If possible, verify translation by examining the enzyme activity. · If endogenous biotinylated protein can be detected but not the protein of interest, too few lysine residues may be in the target protein. Promega Corporation · 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 TB182 · Revised 8/21 6. Troubleshooting (continued) Symptoms Signal development is weak Causes and Comments Insufficient amount of target protein present on the gel. Increase the amount of lysate loaded onto the gel or increase the amount of TranscendTM tRNA used in the reaction. Reagents used were too cold. The colormetric reagent (Western Blue® substrate) must be at room temperature before using. Too much background Too much lysate loaded onto the gel. Decrease the amount of lysate loaded onto the gel. Insufficient blocking or washing. Make sure the protocol is followed correctly. 7. References 1. Johnson, A.E. et al. (1976) N-epsilon-acetyllysine transfer ribonucleic acid: A biologically active analogue of aminoacyl transfer ribonucleic acids. Biochemistry 15, 56975. 2. Kurzchalia, T.V. et al. (1988) tRNA-mediated labeling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products. Eur. J. Biochem. 172, 6638. 3. Beckler, G.S. and Hurst, R. (1993) tRNAnscendTM non-radioactive detection of in vitro translation products labeled using biotinylated lysine tRNA. Promega Notes 43, 2431. 4. Dayhoff, M.O. (1978) Atlas of Protein Sequence and Structure, Suppl. 2, National Biomedical Research Foundation, Washington. 5. TNT® Coupled Reticulocyte Lysate Systems Technical Bulletin #TB126, Promega Corporation. 6. Crowley, K.S., Reinhart, G.D. and Johnson, A.E. (1993) The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell 73, 110115. 7. Luciferase Assay System Technical Bulletin #TB281, Promega Corporation. 8. CAT Enzyme Assay System With Reporter Lysis Buffer Technical Bulletin #TB084, Promega Corporation. 9. Galacto-LightTM -Galactosidase Reporter Assay System Instruction Manual, Tropix, Inc. 12 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 TB182 · Revised 8/21 www.promega.com 8. Appendix 8.A. Composition of Buffers and Solutions Tris-buffered saline (TBS) 20mM Tris-HCl (pH 7.5) 150mM NaCl TBS + 0.5% Tween® 20 (TBST) 20mM Tris-HCl (pH 7.5) 150mM NaCl 0.5% Tween® 20 SDS polyacrylamide running 10X buffer 30g Tris base 144g glycine 100ml 10% SDS Add water to a final volume of 1L. 8.B. Related Products Eukaryotic Transcription/Translation Systems Product TNT® SP6 High-Yield Protein Expression System TNT® T7 Quick Coupled Transcription/Translation System TNT® SP6 Quick Coupled Transcription/Translation System TNT® T3 Coupled Reticulocyte Lysate System TNT® T7 Coupled Reticulocyte Lysate System TNT® SP6 Coupled Reticulocyte Lysate System TNT® T7/SP6 Coupled Reticulocyte Lysate System TNT® T7/T3 Coupled Reticulocyte Lysate System TNT® T7 Quick for PCR DNA E. coli S30 Extract Systems Product E. coli S30 Extract System for Circular DNA E. coli S30 Extract System for Linear DNA E. coli T7 S30 Extract System for Circular DNA Size 40 × 50l reactions 10 × 50l reactions 40 reactions 40 reactions 40 reactions 40 reactions 40 reactions 20 reactions of each 20 reactions of each 40 reactions Size 30 reactions 30 reactions 30 reactions Cat.# L3260 L3261 L1170 L2080 L4950 L4610 L4600 L5020 L5010 L5540 Cat.# L1020 L1030 L1130 Promega Corporation · 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 TB182 · Revised 8/21 8.B. Related Products (continued) Rabbit Reticulocyte Lysate Translation Systems Product Rabbit Reticulocyte Lysate, Nuclease Treated Flexi® Rabbit Reticulocyte Lysate System Size Cat.# 1ml L4960 1ml L4540 Rabbit Reticulocyte Lysate/Wheat Germ Extract Combination System Product Rabbit Reticulocyte Lysate/Wheat Germ Extract Combination System Size 12 reactions Cat.# L4330 Available Separately Product Streptavidin Alkaline Phosphatase Western Blue® Stabilized Substrate for Alkaline Phosphatase Amino Acid Mixture, Complete Size 0.5ml 100ml 175l Cat.# V5591 S3841 L4461 8.C. Summary of Changes The following changes were made to the 8/21 revision of this document: 1. Removed discontinued Cat.# L5080 and associated chemiluminescent detection instructions (Section 5.D). 2. Updated Sections 1 and 6, including Figure 2. 3. Moved to a new template. © 20112021 Promega Corporation. All Rights Reserved. Flexi, RNasin, TNT and Western Blue are registered trademarks of Promega Corporation. Transcend is a trademark of Promega Corporation. AMBIS is a trademark of AMBIS, Inc. Coomassie is a registered trademark of Imperial Chemical Industries, Ltd. Corning is a registered trademark of Corning, Inc. Galacto-Light is a trademark of Tropix, Inc. Kodak is a registered trademark of Eastman Kodak Co. Tween is a registered trademark of ICI Americas, Inc. 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. 14 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 TB182 · Revised 8/21 www.promega.comAdobe PDF Library 15.0