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IND-4. Development of the Immune System and the Western Press-Blot. In most mammals, antibody production does not occur until after birth. The newborn calf receives antibodies in milk and these antibodies are responsible for passive immunity during early postnatal life. Thus, fetal calf serum is devoid of antibodies, neonatal calf serum contains antibodies of maternal origin while serum from the adult contains large amounts of antibodies made by the mature immune system. This fascinating developmental scheme is illustrated in this exercise. First, students electrophorese serum proteins from fetal, newborn, and adult animals on an agarose gel and the separated proteins are transferred onto a nitrocellulose membrane by the Western Press-Blot procedure shown on the facing page. All proteins on the blot are stained with protein blot stain and then the major class of circulating antibodies (IgG) is detected by using an immunological procedure. The results show that albumin and transferrin are present in sera from all stages while the antibodies are absent in fetal serum but present at high levels in serum from the adult. This analysis can be performed during a single 3-hour, or two 2-hour laboratory sessions. The exercise was designed for 8 groups of students and includes: fetal calf serum, newborn calf serum, adult cow serum, anti-Cow IgG (peroxidase linked), nitrocellulose, blotting paper, protein blot stain, Tris buffer saline (TBS), TBS + NP40, color development buffer, chloronapthol, hydrogen peroxide, dishes for blot incubation, and gelatin. The PROCELL Electrophoresis Unit and Electrophoresis Package 1/8 are needed but not provided.
IND-5. Specificity of Albumin Binding. The binding of an enzyme to its substrate is only one example of the many specific molecular interactions that occur in biological systems. An analogous binding process occurs with serum albumin which binds certain small molecular weight compounds and serves as a carrier molecule for these compounds in blood. In this exercise, students use an electrophoretic assay to examine the binding of various dyes to albumin. The results of this graphic analysis show that the binding of dyes to albumin is saturable, specific, compatible, and dependent on the native structure of the protein. The exercise is designed such that each of the eight groups of students performs a different experiment. Each group then describes their results and conclusion to the entire class. This exercise is a valuable experience in analyzing data and provides a fine introduction to enzyme kinetics. The analysis can be performed during a single 3-hour or two 2-hour laboratory sessions and includes: bovine serum albumin, dilution buffer, bromophenol blue, orange G, ponceau S, sodium dodecyl sulfate, and rabbit hemoglobin. Electrophoresis Package 1/8 is needed but not provided.
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