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This program stresses the relevance of molecular biology to the study of physiology and human disease. Students first learn how proteins are separated on the basis of charge by electrophoresis. With this information, they perform a series of experiments aimed at illustrating the importance of the technique in biology and medicine. Students examine proteins in biological fluids and relate their findings to physiological and pathological states. They also study specific antigens and enzymes in serum and the abnormal hemoglobin in patients suffering from sickle cell anemia. This fine collection of experiments provides an introduction to electrophoresis and to protein composition, structure, and function in health and disease. 101. Electrophoretic Separation of Proteins
Students are introduced to the theory of separating proteins according to charge differences using electrophoresis. They then study four proteins and relate differences in their charges to their migration rates in an electric field. Each protein is a different color so that its progress during the separation can easily be followed. 102. Genetics and Sickle Cell Anemia Many changes in the structure of hemoglobin have arisen by mutations. About one person in 100 carries a mutant hemoglobin gene, and these individuals have abnormal hemoglobin molecules in their blood. One of the most common abnormal hemoglobins is hemoglobin S, which causes sickle cell anemia. When the gene for hemoglobin S is inherited from both parents, all of the hemoglobin in the circulation is hemoglobin S and the individual suffers from severe anemia. When the gene for hemoglobin S is inherited from only one parent, the individual is heterozygous for the condition and has sickle cell trait. Although these individuals rarely have severe anemia, half of their hemoglobin is hemoglobin S and half is normal hemoglobin. In this exercise, students compare the electrophoretic patterns of hemoglobin from a normal individual to hemoglobin S and hemoglobin from a person with sickle cell trait. Students are also given the opportunity to study their own hemoglobin in order to test for hemoglobin variants. Typical results of this exercise are shown below. Normal and Sickle Hemoglobin EXP 102 1 2 3 4 5 6 7 8
In this analysis, hemoglobin from a normal individual (gel lanes 1, 4, 5, 8 ) is compared to hemoglobin S (lanes 3 & 7) and hemoglobin from a person who has sickle cell trait (lanes 2, & 6). Note the two forms of hemoglobin in the individual with sickle cell trait.
103. Analysis of Serum Proteins Serum contains a variety of small molecules, as well as hundreds of different serum proteins. Serum proteins are frequently separated and characterized by electrophoresis in the clinical laboratory to determine the concentration of various proteins and to detect abnormal protein species. In this exercise, students use electrophoresis to study cow serum and to identify major serum proteins including albumin, transferrin and antibodies. This exercise serves as a good introduction to the function and complexity of proteins in blood.104. Evolution of Serum Proteins Antibodies can be used to study evolution at the molecular level. In this exercise, immuno-electrophoresis and double diffusion are used to illustrate this important concept. The analysis shows that cow serum proteins are similar to those from sheep and goat but are distinct from serum proteins from horse, donkey and chicken. This exercise acquaints students with basic immunological techniques and serves as an excellent illustration of the utility of these techniques for the study of protein evolution. This exercise requires two 2-3 hour laboratory periods. 105. A Comparison of Human and Bacterial Amylase A powerful technique used in the modern laboratory involves the detection of specific enzymes in agarose gels following electrophoresis of proteins from serum, tissue extracts and other biological preparations. This procedure makes it possible to study certain properties of individual enzymes in complex protein mixtures. In this exercise, students are introduced to this procedure by comparing the amylase found in bacteria with that in their own saliva. 106. Protein Fingerprinting A comparison of specific proteins from different species provides a powerful approach for establishing evolutionary relationships and for identifying organisms. A common approach used for this purpose is protein fingerprinting where electrophoretic properties of specific proteins are analyzed in different species. In this exercise, students use the approach to compare the forms of the enzyme lactate dehydrogenase that are found in serum of different mammals. Typical results of this graphic experiment are shown below. The results illustrate that each species has a characteristic pattern of bands and that the pattern in sheep and goat is similar while the patterns from cow and especially horse are distinct. Forms of lactate dehydrogenase in serum
Serum proteins from cow (lanes 1, 5), sheep (lanes 2, 6), goat (lanes 3, 7), and horse (lanes 4, 8) were separated by electrophoresis. The different forms of lactate dehydrogenase were then detected by activity staining
Click here for the Contents of the Chemical Package
Individual Experiments Select the individual experiments in this series that best suit your needs. Each experiment is supplied with the chemicals needed for 16 students working in pairs. Each one also comes with an instructor guide and a single student guide which you may reproduce for educational purposes. Student guides give background information and detailed instructions that are necessary for successful performance and comprehension of the experiments. The instructor guide illustrates the results of the exercise and gives answers to the study questions posed in the student guide. If you chose one or more of the experiments below, you should also order Electrophoresis Package 1/8. This Electrophoresis Package provides sufficient agarose, gel stain and electrophoresis buffers for up to 6 of the individual experiments in this series. Note that Electrophoresis Package 1/8 is also suitable for the experiments in Standard Program 8 and Experiments IND-4, IND-5 and IND-24.
The following experiments include chemicals and instruction guides.
Individual Instruction Guides Individual instruction guides for Experiments 101-106, 201-206, 301-306, 401-403, 701-703, 801-804, 1001-1004 and IND-2 thru 23 may be purchased at a price of $3.39 each. Please specify the Experiment title and number on your order.
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