isolation of ribosomes and electrophoresis of ribosomal components
Transport processes are based on the movement of matter when a force is exerted upon them (1). Diffusion, centrifugation, and electrophoresis are all based on forces causing movement or flow of particles. Diffusion is the migration of particles based on a concentration gradient. Sedimentation and electrophoresis are two transport processed in which the force exerted serves to separate particles. Sedimentation depends upon an increase in centrifugal force to cause a flow of matter. Electrophoresis is performed with an electrical field to provide the force necessary to cause the migration of particles through a medium. 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
All materials and methods used are described in the University of Victoria Biochemistry 301 Lab Manual 2000-2001, (1). Instead of using 5ul. Of 28S/18S rRNA in part D, 5ul. of 23S/16S rRNA standard was used. Also, in the seperation of proteins by SDS-PAGE , a current of 14mA was applied to the stacking gel, and a current of 28mA was applied to the resolving gel. 5. Sikorski, M., Przbl, D., and Legocki, A.,(1979). Pant Science Letters, 15, 387-397. 1. Biochemistry 301 Laboratory Manual, 2000-2001. University of Victoria. Victoria, B.C. 9-1 to 9-15, A-1 to A-7. The 0.8% agarose gel, shown in Figure 2, depicts 70S and 80S ribosomal rRNA and two rRNA standards. A 23S/16S rRNA standard, which is in lane 4, produced two bands observed under ultraviolet light. The 5S rRNA standard in lane 5 gave only one band, which was the furthest from the origin. The 70S ribosome rRNA extract, shown in lane 5-7, resulted in three distinct bands, corresponding to the 5S, 16S, and the 23S bands. The 80S extract, in lanes 1-3, also gave these three bands, with a slight shift. However, an additional fourth band was observed between the 5S and 16S Bands in all threee lanes, probably the case of broken down product. The SDS-PAGE of the 80S ribosome protein extract from wheat germ embryol showed 29 bands, as seen in Figure 1. These proteins ranged from 12589 Da to 100000 Da. The concentration of bands was at the lower end of this molecular weight range. Data by sikorski, et al. , corresponds to this observation, stating that the average molecular weight is 21,400 Da and an overall range from 11,500 to 41,800 Da (5). The bands observed above this range are likely due to the same reasons described above. A large discrepancy exists between the observed 29 bands and the reported 82 different proteins form the wheat germ ribosome. This is likely due to resolution of the gel and overlapping of proteins. Furthermore, the concentration of protein in the sample may have been to low to observe some bands. This could be caused by inadequate resuspension of the protein pellet prior to loading the sample. ------------------------------------------------------------------------ ds will be used in both gels, and results will be compared to literature data. The SDS-PAGE gel in Figure 1 shows the separation of 70S and 80S ribosomal proteins, in comparison with a BioRAD standard. The 70S protein extract, shown in lanes 5-7, had a total of 32 bands,with molecular weights ranging from Da to Da. The concentration of these bands is at the higher end of this molecular weight range. The 80S ribosomal extract, in lanes 1-3. There were 30 bands observered in lane 8, ranging from Da to Da. The majority of these bands were concentrated in the lower end of this molecular weight range. Differential centrifugation is based on the fact that different proteins have varying masses and densities which will settle at different levels due to an equilibration between centrifugal force which is directly dependen
Some common words found in the essay are:
Da Da, Data Wittmann, SDS-PAGE Specifically, SDS-PAGE Sodium, Introduction Transport, Treatment SDS, Lab Manual, Results SDS-PAGE, Rev Biochem, Ecoli Mre, molecular weight, nucleic acids, protein extract, agarose gel, rrna standard, weight range, bands observed, 5s 16s, molecular weight range, 16s 23s, 23s/16s rrna standard, figure 2, 5s 16s 23s, 5s rrna standard, ranging da da,
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