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  Section: Plant Lab Protocols
 
 
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Methodology for Amino Acids and Proteins

 
     
 
Preparation of S-30 extract for protein synthesis in vitro

Protein synthesis is a complex biosynthesis reaction involving a large number of cell components and molecules. The process takes place on ribosomes and involves polymerization of amino acids at the expense of energy as directed by messenger ribonucleic acid. The protein synthesis as takes place in vivo could be conducted in vitro using cell-free extracts. These extracts are prepared from a variety of sources such as reticulocyte, wheat embryo, wheat germ etc. These extracts are very useful tools to study the synthesis of protein products programmed with exogenous mRNAs. This procedure describes a method to prepare an efficient cell-free extract from wheat embryos/germs.


Principle
The starting material is extracted in a suitable buffer to release the cell content, centrifuged to get rid of fat and mitochondria and the post-mitochondrial supernatant containing ribosomes and soluble components is used for protein synthesis in vitro.




Materials
Wheat Embryo/Wheat Germ
Standard HEPES Buffer (SHB)
    20mM HEPES-KOH (pH 7.6)
    120mM KC1

    2mM Magnesium Acetate
    6mM 2-Mercaptoethanol
SHB (pH 6.25)
 
 
Procedure
1.
Prechill a mortar and pestle by adding a few mL of liquid nitrogen. Add 3g of wheat embryo/wheat germ when liquid nitrogen is still there. Grind to a fine powder before thawing. Add 20mL of SHB (pH 6.25) in increments and continue grinding to get a fine homogenate.
2.
Centrifuge the homogenate at 17,000rpm (30,000g) for 10 min in a refrigerated centrifuge.
3.
Remove the supernatant carefully using a Pasteur pipette avoiding both the precipitate and the top lipid layer.
4.
Repeat the steps 2 and 3.
5.
Load the supernatant onto a Sephadex G25 column (25 X 1cm) preequilibrated with SHB (pH 7.6) and elute with the same buffer.
6.
Collect the eluant in fraction. Combine the most turbid fractions. Immediately, dilute 2mL of the combined fraction to 2mL with water and measure the absorbance. The A260/A280 should be above 1.6.
7.
Freeze immediately and store the combined fractions in small aliquots (0.2-0.5mL) in microfuge tubes under liquid nitrogen (-190°C) or at -70°C.
 


 
Notes
1.
Embryos can be collected from freshly harvested wheat grains.  Grind the grains in a coffee mill at full speed twice, each one min duration to break the grains partly. Collect the intact embryos cleaning by air-flow and sieving. Final purification of embryos is carried out briefly by floating in a mixture of organic solvents - cyclohexane: carbon tetrachloride (1: 4) – change the solvent ratio slightly to float the embryos on the surface. Collect the floating embyros quickly and dry on filter paper. Embryos can be stored in sealed vials in freezers for a few weeks before extraction.
2.
Wheat germ may be collected from flour mills Make sure that it has not been heat treated. Clean by flotation as described above.
3.
Wash the pestle and mortar with chromic acid overnight and rinse extensively with water before use.
4.
All operations should be done at 0-4°C.
5.
Extraction at low pH (6.25) buffer helps lower the endogenous activity of the extract.
6.
Endogenous activity can also be reduced by preincubation of the extract with energy mix prior to in vitro translation assay.
7.
The translation efficiency of the extract will be lower if A260/A280 is below 1.6. Carefully combine only peak turbid fractions.
8.
Storing of the extract at -20°C leads up to 70% activity loss in three weeks.
9.
Use only once of the thawed extract.
10.
Embryonic axes of legumes are not good sources of extract for in vitro translation assays.
 
References
1. Carlier, A, Peumans, W J and Manickam, A (1978) Plant Sci Lett 11 207-216.
2. Marcus, A, Efron, D and Weeks, D P (1974) Method Enzymol 30F 94-101.
3. Roberts, B E and Paterson, E M (1973) Proc NatlAcad Sci USA 70 2330.

 
     
 
 
     




     
 
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