The purpose of this study is to use the transport processes of sedimentation and electrophoresis to isolate and examine certain components of prokaryotic and eukaryotic ribosomes. Using differential centrifugation, 70S E.coli Mre 600 ribosomes and 80S wheat germ ribosomes will be isolated. A protein extract from these ribosomes will be obtained by acetic acid/acetone precipitation and analyzed by a SDS-PAGE (Sodium dodecylsulfate polyacrylamide gel). A CHCl3 extraction method will be employed to isolate the RNA components of the ribosomes, which will be analyzed on an agarose gel. Standar

ds will be used in both gels, and results will be compared to literature data.

The protein extract from the 70S E. Coli ribosome showed 32 bands ranging from molecular weights of 10471 Da to 105925 Da when separated using SDS-PAGE, as shown in Figure 1. These bands represent 32 individual proteins. These proteins were concentrated in the middle to higher end of this molecular weight range. Data by Wittmann shows the presence of 53 Proteins present in the 70S E. Coli ribosome, with a range of 5381 to 60159 Da (2). The large variability in the upper limit of this range could be due to a number of factors. Primarily, contamination could be present in the sample, resulting in high molecular weight bands. Another possibility is insufficient cleavage or disulfide bonds by the mercaptoethanol in the sample buffer. This would result in polymeric proteins, thereby giving unusually high molecular weight bands. The difference between the 32 bands observed and the 53 bands reported in literature can be explained by poor resolution of the 1-D gel suffers f!

In the 70S rRNA sample run on a 0.8% agarose gel, three subunits were observed, as in Figure 2. These subunits correspond to the sample markers of 5S, 16S and 23S rRNA. The literature states similar findings in that Sittmann observed components at 5S, 16S and 23S (2).

1. Biochemistry 301 Laboratory Manual, 2000-2001. University of Victoria. Victoria, B.C. 9-1 to 9-15, A-1 to A-7.

Four RNA bands were observed in the 80S ribosome on the agarose gel in Figure 2. Their approximate sedimentation numbers were 5S, 10S, 16S, and 23S. The corresponding reported values, determined by Wool, are 5S, 5.8S, 18S, and 23S for the four bands (6). The reported 5.8S band was not observed due to crowding on the gel and limitations of resolution. The additional band observed at 10S was quite faint and could be due to the degradation of the RNA caused by prolong storage of the samples or Rnase contamination. This degradation accounts for the observed smearing in the photograph.

6. Wool, I.G., (1979). Ann. Rev. Biochem. 48, 719-754.

The protein extraction procedure involved the addition of 0.10 volumes magnesium chloride and two volumes of glacial acetic acid. The sample was then kept on ice for forty five minutes. This separated the protein from the ribosome extract. The magnesium chloride served to increase the yield of protein, and, in conjunction with acetic acid, provided a stable environment for the protein. The upper supernatant with the magnesium ions contained the proteins, which was approximately ninety percent of the original protein in the isolated ribosomes (3). Acetone was added to the organic layer to desolubilize and dehydrate the proteins and cause them to precipitate. Urea, which is a chaotropic agent, denatures proteins by allowing water molecules to solvate non-polar groups in the interior of proteins (4).

All materials and methods used are described in the University of Victoria Biochemistry 301 Lab Manual 2000-2001, (1). Instead of using 5ul

Some common words found in the essay are:
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