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MOLECULAR BIOLOGY: WORKING WITH PROTEINS
PROTEIN EXPRESSION: In Vitro Translation
PREPARATION OF MESSENGER-DEPENDENT RABBIT RETICULOCTYE LYSATE
Preparation of Messenger-Dependent Rabbit Reticulocyte Lysate
To test that the lysate is working before doing the Nuclease inactivation, proceed with the Cell-Free Translation of RNA in the Messenger-Dependent Rabbit Reticulocyte Lysate protocol after step 3. There is no need to add mRNA as endogenous message will give a large incorporation of amino acids into protein. Procedure
1. Thaw 1 ml of lysate (see Hint #1).
2. Add 10 μl of Creatine Kinase Solution and 25 μl 1 mM Haemin.
3. To 800 μl of the supplemented lysate, add 150 μl master mix and mix well.
4. Add 10 μl of 0.1 M CaCl2 and 10 μl of Microccocal Nuclease Solution (about 150 Units)
5. Mix thoroughly and transfer from ice to a ddH2O bath at 20°C for 15 min.
6. Add 20 μl of 0.1 M EGTA and rapidly chill the sample.
7. Divide the messenger-dependent lysate into 50 to 100 μl aliquots and freeze in Liquid Nitrogen.
0.1 M EGTA, neutralized with KOH Micrococcal Nuclease Solution 1 mg/ml in ddH2O 0.1 M CaCl2 SOLUTIONS (List Solutions below) Creatine Kinase Solution
Store at -20°C
5 mg/ml Creatine Kinase in 50% aqueous Glycerol
Amino Acid Mixture Use stock amino acid solutions of 100 mM. Mix equal volumes of the 19 non-radioactive amino acids. Do not add the amino acid that will be used as the label. Master mix 0.2 M Creatine Phosphate
10 mM MgCl2
333 ul/ml Amino Acid Mixture
2 M KCl
1 mM Haemin 13.26 mg Haemin dissolved in 0.4 ml 0.2 M KOH. Add 0.475 ml ddH2O, 0.125 ml 2 M KCl and 4.0 ml Ethylene Glycol, Centrifuge 5 min at 5000 rpm, Store at -20°C. BioReagents and Chemicals
1. Perform all steps on ice except where noted.
Citation and/or Web Resources
Pelham, H and Jackson, R. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem 1976; 67:247-56