Chapter 24
Protein Techniques
ROBERT F. REILLY
Subprotocol 1
ln Vitro Translation and lmmunoprecipitation
Cell-free translation systems contain all the components necessary for protein synthesis: tRNAs; ribosomes; amino acids; initiation, elongation and
termination factors; GTP; ATP; and magnesium. They require only the addition of a transcript tobe translated. Single-stranded RNA for translation
can be transcribed from a wide variety of plasmid vectors, such as pßluescript®, which contain a multiple cloning site downstream from a powerful
promoter for a bacteriophage DNA-dependent RNA polymerase. The two
most common cell-free translation systems in use are the rabbit reticulocyte
lysate system, and the wheat germ extract system. Of these the rabbit reticulocyte lysate system is more commonly used. The major drawback
of the wheat germ system is its tendency to produce incomplete translation
products due to premature termination (Shih and Kaesberg 1976). As a result, it is generally unsuitable for producing polypeptides greater than 60
kDa. One advantage of the wheat germ systemisthat it does not require the
removal ofendogenaus mRNAs. Therefore, it may translate weakly-initiating mRNAs more efficiently than the reticulocyte lysate system. Recent
modifications of the wheat germ system have made it more suitable for the
translation of higher molecular weight proteins (March et al. 1986).
Robert F. Reilly, University of Colorado Health Science Center, Division ofNephrology,
4200 East Ninth Avenue, DenverCO, 80262, USA (phone +0 1-303-399-8020 ext. 31 01;fax
+01-303-393-4611; e-mail Robert.Reilly@uchsc.edu)
Protein Techniques
ROBERT F. REILLY
Subprotocol 1
ln Vitro Translation and lmmunoprecipitation
Cell-free translation systems contain all the components necessary for protein synthesis: tRNAs; ribosomes; amino acids; initiation, elongation and
termination factors; GTP; ATP; and magnesium. They require only the addition of a transcript tobe translated. Single-stranded RNA for translation
can be transcribed from a wide variety of plasmid vectors, such as pßluescript®, which contain a multiple cloning site downstream from a powerful
promoter for a bacteriophage DNA-dependent RNA polymerase. The two
most common cell-free translation systems in use are the rabbit reticulocyte
lysate system, and the wheat germ extract system. Of these the rabbit reticulocyte lysate system is more commonly used. The major drawback
of the wheat germ system is its tendency to produce incomplete translation
products due to premature termination (Shih and Kaesberg 1976). As a result, it is generally unsuitable for producing polypeptides greater than 60
kDa. One advantage of the wheat germ systemisthat it does not require the
removal ofendogenaus mRNAs. Therefore, it may translate weakly-initiating mRNAs more efficiently than the reticulocyte lysate system. Recent
modifications of the wheat germ system have made it more suitable for the
translation of higher molecular weight proteins (March et al. 1986).
Robert F. Reilly, University of Colorado Health Science Center, Division ofNephrology,
4200 East Ninth Avenue, DenverCO, 80262, USA (phone +0 1-303-399-8020 ext. 31 01;fax
+01-303-393-4611; e-mail Robert.Reilly@uchsc.edu)