The mRNA extracted from mammalian cells or produced by in vitro transcription of cloned DNA can be translated in cell-free extracts to synthesize proteins, which can be used in immunoprecipitation assays or for biological activity determination.
Operation method
Translation of mRNA in reticulocyte lysates
Materials and Instruments
Rabbit Pancreatic Ribonuclease Chlorella Ribonuclease Type I Phosphocreatine Kinase Move I. Materials and equipment Caveat 1) The translation activity of reticulocyte lysate depends on the addition of exogenous mRNA. chlorella nuclease is active in the presence of calcium ions, but its digestive activity is lost when EGTA is added. Therefore, it is important to make sure that the endogenous mRNA in the reticulocyte lysate is digested completely to avoid the generation of stray bands during translation, which may affect the results.2) Chloroferricitrin is a strong Artist inhibitor of cIF-2 inhibitor, if lack of chloroferricitrin, the protein synthesis in reticulocyte lysate will be stopped after a short incubation time. Therefore, K should be titrated in the range of 20~80umol/L to obtain the best translation conditions.3) Melt the reticulocyte lysate gently and return the unused lysate to 70℃ or liquid nitrogen as soon as possible.4) The concentration of magnesium acetate in the translation reaction depends on the reticulocyte lysate and the mRNA. The magnesium acetate should be titrated in the range of 0~1 mmol/L, and then the optimal concentration of magnesium acetate should be chosen to determine the effect of different concentrations of potassium chloride (20~8OmmoI/L) on the mRNA for specific purposes. In general, about 70 mol/L of KC1 is most suitable for the translation of capped mRNAs, while for uncapped mRNAs, the optimal concentration is about 40 mmol/L. 5) In the reaction system, KC1 can be added to the reaction system.5) Up to 10ug of total cytoplasmic RNA or 0.2ug of poly(A)+mRNA or in vitro synthesized RNA can be added to the system until saturation. The results obtained with poly(A)+mRNA are less background than with cytoplasmic RNA.6) Addition of saturating amounts of mRNA to the translation system can stimulate the incorporation of [35S] methionine into the acid-precipitated material by up to 10 to 25 times. Under optimal conditions, 25u1 of the reaction mixture contains approximately 3X106〜to 5X106Counts/min (l ~ 3uCi) of [35S] methanethioic acid7) Prior to precipitation of Emmy-tagged proteins with 10% trichloroacetic acid (TCA), treat the reaction solution by incubating the reaction solution with 0.3moI/LNaOH at 37°C for 15 min or heating with 5% to 10% TCA at 100°C for 15 min to disrupt [ 35S].35After precipitating a small amount of protein with TCA, protein sprouts can be detected [ 3535 S] methionine-tRNA.S] methionine incorporation can be measured by precipitating a small amount of protein with TCA.8) Background bands may appear when analyzing reticulocyte lysates by SDS polypropylene amide gel electrophoresis. Radiolabeling of peptides that migrate at an apparent molecular mass of 45 kDa, as well as labeling of beads, is often caused by impurities in the thionine. This problem can usually be avoided by using freshly purchased radiolabeled methionine.9) Double-stranded DNA templates often produce a certain amount of double-stranded RNA during in vitro transcription, which will inhibit oceanization in reticulocytes. If flipping is not efficient, the amount of double-stranded RNA in in vitro transcription should be minimized, and control RNAs known to be active in reticulocytes4 should be mixed with bovine RNA from different aseptic in vitro transcription systems.Translation assays were performed. For more product details, please visit Aladdin Scientific website.
Acetylphenylhydrazine Solution A Solution B Sterilized redistilled water CaCl2 EGTA Chloroferricyclohexine Storage Solution Spermidine Phosphocreatine Amino acids DTT HEPES Water KCl Magnesium acetate [35S] Methionine.
Electrophoresis unit Cheese wrap Centrifuge
(I) Preparation of rabbit reticulocyte lysate
1) 6 rabbits of 2~3 kg.
2) 1.2% Acetylphenylhydrazine, neutralized with 1moL/LHEPES (pH7.0) to PH7.5.
3) Solution A: 140 mmol/L NaCL, l.5 mmol/L magnesium acetate, 5 mmol/L KCL, 0.001% heparin.
4) Solution B: 140 mmol/L NaCl, 1.5 mmol/L Mg acetate, 5 mmol/L KC1, 0.001% heparin.
5) Sterilized redistilled water
6) lOOmmol/LCaCl2
7)200 mmol/LEGTA(pH7.0)
8) Chlorella nucleic acid enzyme (150,000 units/ml), stored in goblet acid concentration of 50 mmol/L (pH 9.2), CaCl2 concentration of 5 mmol/L.
9) Type I phosphocreatine kinase 40n^/ml, stored in 50% glycerol)
10) Chloroferrioxetin storage solution dissolved in ethylene glycol at a concentration of 5 mg/ml), chloroferrioxetin can be prepared as follows: dissolve chloroferrioxetin (molecular halo 652Da) in 400ul0.2mol/LKOH, add slowly in several times, adding a small amount each time and oscillating between each time; add 600ul of water, add lmol/LTris-Cl( pH7.8), 100ul of water, and add 1mol/LTris-Cl( pH7.8), and add 1mol/LTris-Cl( pH7.8). Add 600ul of water, add lmol/LTris-Cl(pH7.8),100ul, adjust the pH to 7.8 by adding 0.1mol/LHC1 dropwise; add 4.8 ml of ethylene glycol, and mix with shaking; centrifuge at 5000 g at 4°C to remove the insoluble precipitate; dilute chloroferrile hemoglobin with lOmmol/LKCl at the ratio of 1:100, and read absorbance at the wavelength of 540nm to calculate the concentration of the chloroferrile hemoglobin (1 mmol/L chloroferrile hemoglobin solution, 1:100). absorbance at 540nm to estimate its concentration (1 mmol/L chloroferrioxamine solution has an absorbance value of 11:1); adjust the concentration of chloroferrioxamine to 5 mg/ml with 4:1 ethylene glycol:water solution.
11) Cheese wrapping.
12) Centrifuge.
(ii) Translation of reticulocyte lysate
1) Translation reaction mixture: Prepare the translation reaction mixture according to the K-method, dispense 50~100ul/small portion, and store at 70℃ or in liquid nitrogen.
100 mmmol/L spermidine 200ul
800 mmol/L phosphocreatine 400ul
5 mmoi/L Amino acid (not methionine) 200ul
lmol/LDTT 80ul
500 mmol/LHEPES (pH 7.4) 1600ul
Water 720ul
5 mm0l/L Amino Acids is a solution containing all amino acids except methionine at a concentration of 5 mmol/L. This solution can be used for radiolabeling proteins synthesized in vitro with [35S ] methionine; if proteins are to be labeled with other radiolabeled amino acids, the appropriate labeling amino acid mixture should be used.
2) lmol/LKCl
3)16.5 mmol/L magnesium acetate
4)T5S] methionine .8O uCi 100~1200Ci/mmol/L, translation grade
5) Pancreatic ribonuclease, l00 ug/ml dissolved in 50 mmol/L EDTA (pH 8.O)
6) Electrophoresis device.
II. Methods of operation
(I) Preparation of rabbit reticulocyte lysate
(1) Select 6 rabbits weighing 2~3 kg and inject acetylphenylhydrazine solution subcutaneously according to the procedure described above to obtain blood from anemic rabbits.
On the first day, 2.0 ml
1.6 ml on the second day
Third injection 1.2 ml
Day 4 injection 1.6 ml
Day 5 injection 2.0 ml
2) On the seventh and eighth days, blood was collected as follows: first, the rabbit's ears were wiped with an alcohol cotton ball, and then a new blade was used to make an incision in the middle ear vein of the longest sprouting segment of the ear. 50 ml of blood could be collected from each rabbit, and it was collected in 50 ml of cooled solution A. On the seventh and eighth days, the amount of blood collected was appropriate. On the seventh and eighth day, the amount of blood collected was appropriate, and on the ninth day, the animals could be anesthetized with pentobarbital or fentanyl (fcntanyl).
On the ninth day, the animals can be anesthetized with pentobarbital or fentanyl (fcntanyl) and then bled by cardiac puncture (approximately 150 ml of blood collected in 100 ml of solution A).
3) The blood is filtered through cheesecloth and then centrifuged at 2000 ft for 5 min at 4°C to extract the yellowish H-cell surface layer, which is a broad sedimentary band of unevenly densified endocytes, and the deposited reticulocytes and erythrocytes are washed three times with solution B, followed by centrifugation at 5000 g. The blood is collected in 100 ml of solution A. The blood is then centrifuged for 5 min at 2000 ft at 4°C.
4) Measure the volume of deposited cells, mix with an equal volume of pre-cooled bacteriophage distilled water, lysed at 0℃, and centrifuged at 20,000 g for 20 min at 4℃ after Imin.
5) To 100 parts of reticulocyte lysate, add 1 part of chloroferritin and 2 parts of CaCl2 solution and mix well.
6) Add 0.05 portion of cocoon nuclease (150,000 units/ml), mix and incubate at 20℃ for 15 min, and then cool in an ice water bath.
7) Add 1 portion of 200 mmol/L EGTA (pH 7.0), mix well.
8) Add 0.4 fen-creatine phosphokinase (4 Gmg/ml), mix well, and then dispense the treated cell lysate into 250~500 ul/small portion, and store at 70℃ or in liquid nitrogen.
(ii) Translation of reticulocyte lysate
1) RNA or mRNA can be obtained by the method described in the previous section.
2) The standard translation reaction system is composed as follows:
Flipzer reaction mix 2ul
1mol/LKCl 2ul
16.5 mmol/L magnesium acetate 1ul
[32S] Methionine 8ul
Chlorella nuclease-treated reticulocyte lysate 10ul
RNA 2ul
incubate at 30℃ for 1 h.
3) In vitro translation reaction product PT was analyzed by immunoprecipitation and SDS polyacrylamide gel electrophoresis.
4) For SDS polyacrylamide gel electrophoresis, add 0.2 v/v of tryptic ribonuclease to the available sample and incubate at -37°C for 15 mm to hydrolyze the [35S ] methionine-tRNA. 2XSDS gel spiking buffer is added immediately thereafter. Immediately add 2X SDS gel spiking buffer and boil the samples for electrophoresis.
5) Store the unused portion of the translation products at -20°C.