Translation of mRNA in wheat germ extracts
Translation of mRNA in wheat germ extracts
Wheat germ extract in vitro translation system for efficient translation of mRNA in viruses and eukaryotic cells in a heterogeneous cell-free system
Operation method
Translation of mRNA in wheat germ extracts
Materials and Instruments
Spermine or 4.0~8.Ommol L spermine Sarcosine kinase Fresh wheat germ Sand grains Move I. Materials and equipment Caveat 1) The translational activity of the extracts varied with different batches of wheat germ material.2) The exogenous mRNA-stimulated incorporation of radioactive amino acids into translation products in the wheat germ extract system was generally less efficient than in the reticulocyte lysate plants.3) The translational activity of wheat germ extract is particularly sensitive to changes in potassium ion concentration. When the potassium ion concentration was lower than 70 mmol/L, the smaller mRNAs were translated preferentially, while the complete translation of the larger mRNAs required 70 mmol/L or higher concentration of potassium acetate. Under appropriate ionic conditions, peptides as large as 200 kDa can be synthesized. In addition, potassium acetate is preferred because of the inhibitory effect of chloride ions on translation, and any residual potassium ions in the RNA preparation should be thoroughly removed by washing with 70% ethanol.4) Since most of the endogenous amino acids have been removed by SephadexG-25 gel chromatography, depending on the amount of wheat germ extract used, it may be necessary to add amino acids (final concentration of 25uol/L and/or tRNA (final concentration of 58ug/ml) in order to optimize translation activity.5) Addition of spermidine or spermine usually facilitates translation, and is especially necessary for the synthesis of larger polypeptides, probably due to their stabilizing effect on larger mRNA molecules. If spermidine or spermine is omitted, the optimal concentration of magnesium acetate should be increased to 4.0~4.3 mmol/L.6) HEPES is a better buffer than Tris-acetic acid for the in vitro translation system of wheat germ extract. If the latter is used, the optimal concentrations of potassium and magnesium ions need to be changed.7) Some commercial inosine kinases have varying degrees of nuclease contamination, so this must be taken into account if larger amounts of inosine kinase are used.8) Larger mRNAs are heated under for 1 min, then quickly cooled on ice, which improves their translation efficiency in the wheat germ extract system.9) The addition of dog pancreatic microsomal membranes to the plants of the Flipzer reaction system allows the detection of the processing of translation products.10) The process of mRNA-stimulated incorporation of radioactive amino acids into the reevaluated products is linear: there is a delay period of 5 min, followed by a linear t:rise period of dOmin after which the process is fully completed. This system is unstable at temperatures above 30°C, and its maximum activity varies with different batches of wheat germ extract products, which are generally incubated at temperatures between 25 and 30°C. The commonly used incubation temperature is 28°C, while the commonly used incubation temperature is 28°C. The highest activity of this system is found in the wheat germ extract. The incubation temperature is usually between 25 and 30°C. The commonly used incubation temperature is 28°C.11) In order to obtain the highest translational activity, the most suitable reaction conditions should be determined for each batch of wheat germ extract, including the concentration of mRNA, the concentration of potassium and magnesium ions and the incubation temperature, etc., and the concentration of various salts in the column chromatography eluent of wheat germ extract should also be taken into consideration. For more product details, please visit Aladdin Scientific website.
Extraction buffer Equilibration buffer DEFC 10X energy buffer Potassium acetate DTT LHEPES mRNA [3 H] or [35S] labeled L-amino acids Trichloroacetic acid (TCA).
SephatkxG-25 (crude grade) High-temperature oven Low-temperature refrigerator High-speed centrifuge Mantle Electrophoresis unit
(I) Preparation of wheat germ extracts
1) Fresh wheat germ: about 5 g
2) Sand grains: about 5 g
3) Extraction buffer: 20 mmol/LHEPES (pH 7.6), 100 mmol/LKC1, 1 mmol/Lmagnesium acetate, 2 mmol/LCaCl2, 6 mmol/L2-mercaptoethanol
4) Equilibration buffer: 20 mmol/LHEPES (pH7.6), 120 mmol/LKCl, 5 mmol/Lmagnesium acetate, 6 mmol/L2-mercaptoethanol.
5) SephatkxG-25 (crude grade)
6)DEFC
7)High temperature oven
8)Low temperature refrigerator
9)High speed centrifuge
10)mortar and pestle
(ii) Translation of mRNA in wheat germ extracts
1) 10X energy buffer: 10 mmoL/LATP, 20 umol/LGTP, 80 mmol/L phosphoinositide. Potassium nucleoside triphosphate salt should be used and pH adjusted to 7.4 ~ 7.6 with NaOH.
2) 0.5~1.0 mol/L potassium acetate, 25 mmol/L magnesium acetate.
3)20 mmol/LDTT.
4)0.2 mol/L HEPES, pH 7.4~7.6.
5)0.6~1.2 mol/L spermine or 4,0~8.Ommol/L spermidine.
6)mRNA: Obtained as described in the previous section.
7)200~500 ug/ml sarcosine kinase.
8) [3 H]- or [35S ]-labeled L-amino acid: To detect the translation product, an amino acid that has been radiolabeled with 10 to 50 pCi and is abundant in the translation product is added to the reaction system. Suitable specific activities are 140 Ci/mmol for [3 H]-labeled amino acids and 1 Ci/mmol for [35 S]-labeled amino acids. The radioactive amino acids should be in aqueous solution, since ethanol, salts, detergents and some solvents can interfere with translation. Ethanol should be removed by freeze-drying; the effect of other components on translation should be determined by prior analysis. [35S ] Labeled amino acids must be stored at 70°C in small portions so that they are stable for six months, after which time the degradation products, i.e., sulfenyls, may inhibit translation.
9) Trichloroacetic acid (TCA).
10) Electrophoresis device.
II. Method of operation
(I) Preparation of wheat germ extracts
Since the components of wheat embryo in vitro translation system are very sensitive to temperature, they should be stored at one 70°C in appropriate volume portions and minimize the number of freezing and thawing. The whole experimental process should be carried out under sterilized conditions. To prevent RNase contamination, the equipment used should be treated with high temperature (25°C, 8 h) or DEPC, and then washed thoroughly with sterilized distilled water. The preparation should be carried out at 4°C; it is preferable to use plastic containers because the initiation factor tends to stick to glassware.
1) Mix fresh wheat germ (approx. 5 g) with an equal mass of sand grains and gradually add 28 ml of extraction buffer and grind together.
2) The mixture is centrifuged at 28OOOg for 10 min at 2°C, pH 6.5. pH 6.5 such as this prevents the release of endogenous mRNAs from the polysomes, and thus eliminates the need for pre-warming to induce the formation of polysomes.
3) The supernatant is added to a SephadexG-25 gel column equilibrated with equilibration buffer and elution is recovered to remove endogenous aminocaproic acid and phytochromes that inhibit translation. The gel column was then subjected to reverse chromatography to recover amino acids from the gel column.
4) The fractions with a combined well OD260>20 were dispensed at a concentration of OD260 of about 100 and stored at one 70°C. The extracts were then analyzed by reverse chromatography. The extract translation activity can be maintained for more than one year.
(ii) Translation of mRNA in wheat germ extracts
All preparation operations should be carried out on ice. The remaining portion of the zygote fraction after use should be quickly frozen on dry ice. The reaction is carried out in sterilized plastic microcentrifuge tubes
1) Mix the following solutions (up to 60ul total): 5ul of energy mixture, 5ul of potassium/magnesium acetate solution, 5ul of DTT, 5ul of HEPES, 5ul of arginine, 10ul (0.3~8.0ug) of mRNA aqueous solution, 10ul of wheat germ extract, lOul of myoinositide kinase (〇 D260=0.8~1.0), and 5ul of [5 H]or [ 35S] labeled L-amino acid. 35S]-labeled L-amino acids. Creatine kinase should be added last to avoid energy waste. Mix the solution by lightly shaking the centrifuge tube. If desired, microsomal membranes (OD260=0.5) can be added prior to creatine kinase to detect modified processing of translation products.
2) Hold the reaction at 28°C for lh. Finalize at 4°C for lh.
3) Take a portion of the reaction solution and perform TCA precipitation to examine the incorporation of radioactive amino acids into the translation product.
4) The remaining in vitro translation products can be further analyzed by ion exchange chromatography. However, for specific products, immunoprecipitation can be performed first and then analyzed by SDS polyacrylamide gel electrophoresis.