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.
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.
A first step in characterizing a protein often involves determining its molecular weight. From this information, different proteins may be compared and the number of amino acid residues in a protein can be determined. Here, students determine the molecular weight of two unknown proteins by comparing their electrophoretic migration with the migration of standard proteins. The protein standards and unknowns have been pre-stained so that your students can follow their progress during the separation as shown.
A first step in characterizing a protein often involves determining its molecular weight. From this information, different proteins may be compared and the number of amino acid residues in a protein can be determined. Here, students determine the molecular weight of two unknown proteins by comparing their electrophoretic migration with the migration of standard proteins. The protein standards and unknowns have been pre-stained so that your students can follow their
progress during the separation.
SDS gel electrophoresis is used extensively to separate and identify denatured proteins. However, because this method relies on protein size alone, little information about proteins with the same molecular weight can be obtained. Peptide mapping is one of a number of techniques used to study the relatedness of similarly sized proteins. With this method, proteases are used to cut proteins into smaller peptide fragments and the fragments derived from two or more proteins are compared.
SDS gel electrophoresis is used extensively to separate and identify denatured proteins. However, because this method relies on protein size alone, little information about proteins with the same molecular weight can be obtained. Peptide mapping is one of a number of techniques used to study the relatedness of similarly sized proteins. With this method, proteases are used to cut proteins into smaller peptide fragments and the fragments derived from two or more proteins are compared.
The Western blotting procedure is rapidly replacing conventional methods for identifying and characterizing specific proteins in complex protein mixtures. This technique is used extensively for this purpose in the research laboratory and is increasingly being used in diagnostic medicine for detecting proteins of disease agents such as the structural core proteins of the AIDS virus. Here, students will perform this technique to examine the evolutionary distance between different mammals.
The Western blotting procedure is rapidly replacing conventional methods for identifying and characterizing specific proteins in complex protein mixtures. This technique is used extensively for this purpose in the research laboratory and is increasingly being used in diagnostic medicine for detecting proteins of disease agents. Here, students will perform this technique to examine the evolutionary distance between different mammals.
Purified proteins are often needed in the basic research laboratory and for diagnostic and therapeutic procedures. An effective technique for protein purification is affinity chromatography, which exploits a specific interaction between a protein and a complementary binding molecule. In this exercise, students isolate albumin from horse serum by affinity chromatography using a column matrix containing a reactive blue dye which binds specifically to the albumin molecule. They then use electrophoresis to analyze the isolated protein in order to verify the effectiveness of the procedure.
Purified proteins are often needed in the basic research laboratory and for diagnostic and therapeutic procedures. An effective technique for protein purification is affinity chromatography, which exploits a specific interaction between a protein and a complementary binding molecule. In this exercise, students isolate albumin from horse serum by affinity chromatography using a column matrix containing a reactive blue dye, which binds specifically to the albumin molecule. They then use electrophoresis to analyze the isolated protein in order to verify the effectiveness of the procedure.
Chemical signaling between cells in multicellular organisms is frequently mediated by cell-surface receptors. The receptors for neurotransmitters, protein hormones, growth factors, and plant lectins are a few of the many known examples of these important membrane components. In this exercise, students examine the cell location and properties of the receptor for the lectin concanavalin A. In the first experiment of the series, students use a concanavalin A-peroxidase complex in a microscopic assay to show that the specific receptor is found on the surface of their own cheek epithelial cells.
Western blotting is one of the most powerful methods in molecular biology for identifying and characterizing specific proteins in complex protein mixtures. We have now streamlined western-blotting procedures so that the entire analysis can be performed during a single 3-hour, or two 2-hour laboratory sessions. In exercise 801, students use the procedure outlined below to identify albumin, transferrin and gamma globulins in serum and then to study the evolutionary relationships of albumin in vertebrates.
Isoenzymes are different molecular forms of the same enzyme and five major lactate dehydrogenase (LDH) isoenzymes are found in vertebrate tissues. The amounts of the isoenzymes vary in a tissue specific manner and these differences can be readily detected by localizing LDH activity in an agarose gel after electrophoresis of tissue extracts. In this exercise, students prepare a tissue extract from calf thymus and then compare the LDH isoenzyme profile to those from calf serum, heart and muscle.
This miniprogram was designed to give students a basic understanding of enzyme kinetics. In the first experiment in this series, students prepare an extract from wheat germ. They then determined the initial velocity (Vo) of the reaction catalyzed by purified acid phosphatase and by the acid phosphatase activity present in the extract. From these data, they estimate the amount of the enzyme that is present in the wheat germ. In the second experiment, the student examines the effects of substrate concentration on the reaction velocity.
Each protein carries in its amino acid sequence information pertaining to its evolutionary history and origin, and provides clues to the evolutionary history of the organism in which it is found. Indeed, proteins existing today are in effect living fossils. This concept is illustrated in this exercise where eight groups of students examine the abilities of antibodies against cow gamma globulin to react with gamma globulins in the sera of cow, goat, sheep, horse, and chicken.
Microtubules are hollow cylinders made up of polymers of the protein tubulin. Microtubules are major components of cilia and flagella, which are tail like projections that are covered by a plasma membrane and extend outwards from the cell. Motile cilia are used for locomotion and food gathering by some protozoa and are found in the lining of the trachea, where their wave like motion propels mucus, dust and other foreign matter out of the lungs.
The primary level of chromosome structure in eukaryotes occurs when the DNA molecule is wrapped around histone proteins into particles called nucleosomes. Evidence for this “beads on a string” model is derived from nuclease digestion studies. When nuclei are incubated with micrococcal nuclease, the enzyme cleaves the linker DNA between nucleosomes (the string) but not the nucleosomal core DNA (the beads).
The ELISA (enzyme-linked immunosorbant assay) is a powerful immunological method for detecting specific proteins in complex protein mixtures. The ELISA has become an important tool for the cell and molecular biologist. It is increasingly being applied in clinical medicine for detecting proteins associated with disease including antibodies produced in response to infection by the HIV virus. The method is also easy to perform and yields graphic results making it well suited for the teaching laboratory.
The emission of light by living organisms is a fascinating process. The genetic system required for luminescence in the bacterium Photobacterium (Vibiro) fischeri is the lux operon. This operon contains a gene for luciferase (the enzyme that catalyzes the light-emitting reaction) and genes for enzymes which produce the luciferins (which are the substrates for the light-emitting reaction.). In this exercise, students create a luminescent population of bacteria by introducing into E.coli a plasmid that contains this lux operon.