Molecular Techniques and Methods

In vitro Translation using Wheat Germ Extracts

Copy Right © 2001/ Molecular-Informations LLC

INTRODUCTION

An in vitro translation system based on wheat germ extracts has been used for several years. In this procedure, high spermidine concentrations (600 uM) and RNase inhibitors are included in the translation reaction to get the high quality translation products.




MATERIALS AND SOLUTIONS

Floating Solution
Cyclohexane --------------------------------------------------- 50 - 80 ml
Carbon tetrachloride ------------------------------------------- 250 ml


Extraction Solution (100 ml)
10 mM Tris-acetate (pH 7.5) ---------------------------------- 1 ml of 1 M Tris-acetate
3 mM Magnesium acetate -------------------------------------- 0.3 ml of 1 M Magnesium acetate
50 mM Potassium acetate -------------------------------------- 5 ml of 1 M Potassium acetate
Deionized H2O ------------------------------------------------- 93.6 ml
  • Autoclave for 30 minutes.
  • Add 1 mM DTT --------------------------------------------- 0.1 ml of 1 M DTT


    Sephadex G-25 fine (Pharmacia, Sweden)
    Equilibrate with the Extraction Solution


    5 x Energy Mix (1 ml)
    2.5 mM ATP (dipotassium salt, pH 7)
    1.5 mM GTP (trisodium salt, pH 7)
    100 mM Creatine phosphate (dipotassium salt, pH 7)
    250 mg Creatine phosphokinase
    DEPC-treated H2O
  • This mix must be freshly prepared.


    10 x Salt Mix (1 ml)
    200 mM Hepes-KOH (pH 7.5)
    1 M Potassium acetate
    6 mM Spermidine
    2.4 mM each Amino acid except Methionine
    80 uM Methionine
    DEPC-treated H2O
  • This mix can be stored at -20oC for weeks.




    PROCEDURES

    Preparation of the Wheat Germ Extract

    1. Float 30 g of fresh wheat germ (which is not previously heat treated) on a 300 ml of Floating Solution under a hood.

    2. Discard nonfloating materials which is rich in endosperm and not suitable for the preparation of the wheat germ extract.

    3. Recover the floating wheat germ with a sieve.

    4. Repeat steps 1-3.

    5. Dry overnight on a filter paper under a hood.
  • (The fresh wheat germ and the floated wheat germ can be stored dry in sealed bags at 4oC for several months.)


    • 6. Grind 3g of floated wheat germ with 3 g of sterile broken Pasteur pipettes in a precooled mortar. A fine powder should be obtained.

    7. Add 10 ml of Extraction Solution and continue to grind for an additional 30 seconds until a smooth paste results. Keep temperature near 0oC

    8. Centrifuge the homogenate for 10 minutes at 23,000g and 2oC.

    9. Collect the supernatant and centrifuge again for 10 minutes at 23,000g and 2oC.

    10. Load the upper 3/4 of the supernatant (about 4 ml) immediately onto a Sephadex G-25 column (2 X 15 cm) equilibrated with the Extraction Solution.

    11. Collect fractions (1.25 ml) in microfuge tubes as soon as all the extract has penetrated into the column.

    12. Pool the four-most opaque fractions appearing in the effluent (usually fractions 11 to 14 or 12 to 15) in a 15-ml Corex tube.

    13. Leave on ice for 5 minutes to allow high-molecular-weight inhibitor(s) to aggregate.

    14. Centrifuge the homogenate for 10 minutes at 23,000g and 2oC.
  • (Taking care that the temperature always remains close to 0oC, since the aggregate dissolves above 6oC).


    • 15. recover the upper 3 ml and aliquote 100 ul each into microfuge tubes.

    16. Freeze immediately in liquid nitrogen.

    17. Regenerate the Sephadex G-25 column by thoroughly washing with 500 ml of Extraction Solution.

    18. Store wheat germ extract at -70oC.


    In Vitro Translation

    All components should be RNase-free!

    19. Mix the following components in a microfuge tube. The volume of the incubation is 25 ul.

    5 x Energy mix
    5 ul
    10 x Salt Mix
    2.5 ul
    [35S]-Methionine (1000 Ci/mmol; 10 mCi/ml)
    1 ul
    Wheat germ tRNA (3 ug/ul)
    1 ul
    mRNA (5 ug/ul)
    1 ul
    DEPC-treated H2O
    1.5 ul
    RNase inhibitor (25 U/ul)
    1 ul
    Wheat germ extract
    12 ul



    20. Incubate at 25oC for 2 hours.


    Analysis of the Translation Products

    21. Check the radioactivity incorporated into the translation products.
  • Spot 2 ul aliquots onto TCA-treated Whatman 3MM filter discs.

  • Boil the filter discs for 10 minutes in 5% TCA.

  • Rinse the filter discs in 5% TCA.

  • Rinse the filter discs in 5% TCA again.

  • Rinse the filter discs in ethanol.

  • Rinse the filter discs in ethanol: ether (1:1).

  • Rinse the filter discs in ether.

  • Dry the filter discs.

  • Check the radioactivity incorporated by liquid scintillation counter.



    • 22. Analyz the translation products on a 12.5% SDS-PAGE slab gel.
  • Load equal amounts of radioactive material (3-5 X 105 cpm) into each slot of the gel.

  • Electrophorese at 60-80 volts overnight until the bromophenol blue dye has reached the bottom of the 14-cm-long running gel.

  • Dry the gel, and autoradiographed for 2-3 days.





    • NOTES

  • RNase inhibitor is necessary in the in vitro translation to obtain high-molecular-weight translation products in the wheat germ extract.


  • The high spermidine concentration (600 uM) stimulates the efficiency of elongation in this system.





    • KIT INFORMATION




    REFERENCES

  • Goldbach, R.W., Schilthuis, J.G., and Rezelman, G., 1981, Biochem. Biophys. Res.,Commun. 99, 89.


  • Joshi, S., and Haenni, A.L., 1984, FEBS Lett. 177, 163.


  • Laeninih, U.K., 1970, Nature (London) 227, 680.


  • Marcu, K., and Dudock, B., 1974, Nucleic Acids Res. 1, 1385.


  • Mumford, R.A., Pickett, C.B., Zimmem, M., and Strauss, A.W., 1981, Biochem. Biophys. Res. Commun. 103, 565.


  • Roberts, B.E., and Paterson, B.M., 1973, PNAS, 70, 2330.


  • Scheele, G., and Blackburn, P., 1979, PNAS, 76, 4898.


  • Shib, D., and Kaesberg, P., 1976, J. Mol. Biol. 103, 77.


  • Zagorski, W., 1978, Anal. Biochem. 87, 316.

    •
  • Please send your comment on this protocol to "editor@MolecularInfo.com".
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