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PROTEIN EXPRESSION: In Vitro Translation


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.
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
Magnesium Chloride
Potassium Chloride
Calcium Chloride
Ethylene Glycol
Micrococcal Nuclease
Creatine Phosphate
Creatine Kinase
Potassium Hydroxide
Nitrogen, Liquid
Protocol Hints
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