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A common procedure used for determining the size of proteins in the research laboratory is polyacrylamide gel electrophoresis, where denatured proteins are separated in the presence of the detergent sodium dodecyl sulfate (SDS). However, preparing polyacrylamide gels is laborious and requires a number of toxic chemicals. To avoid these limitations, we provide a special blend of nontoxic agarose that yields excellent resolution of SDS-treated proteins. In this innovative series, students use SDS-agarose gel electrophoresis to perform procedures including molecular weight determinations, peptide mapping analysis, detection of specific enzymes in crude cell extracts, affinity chromatography, and immunological studies using the Western blot procedure.
201. Molecular Weight Determination 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 below.
Electrophoresis Time (Minutes) Prestained protein standards of known molecular weights and two proteins of unknown molecular weights were subjected to electrophoresis for the indicated times. The molecular weights of the unknown proteins can be determined by comparing their migration with the migration of the standard proteins. 202. Identifying Sex-Specific Proteins Vitellogenin is a protein produced in hen liver under the influence of estrogen. This sex-specific protein enters the circulatory system and is transported to the ovaries where it is broken down into the egg yolk proteins lipovitellin and phosvitin. In this exercise, students compare the proteins in egg yolk and in hen and rooster sera by electrophoresis and identify vitellogenin, lipovitellin and phosvitin. This exercise illustrates the use of electrophoresis for protein identification and introduces concepts in molecular endocrinology. 203. Comparing Human and Bacterial Amylase Amylase in animals, plants and bacteria can be detected and characterized by the procedure described in this exercise. The analysis is performed by incorporating starch into an SDS-agarose gel prior to electrophoresis of the denatured proteins. After electrophoresis, the SDS is removed and the enzyme spontaneously renatures. The location of amylase in the gel is then carried out by staining the starch in the gel with iodine. Zones of enzyme activity are devoid of starch and are seen as clear bands against a background of blue. Using this procedure, students determine the molecularweight of amylase in their own saliva and compare this value to the molecular weight of amylases produced by bacteria. 204. Peptide Mapping Analysis 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 number and size of fragments generated from a protein are determined largely by the protein’s amino acid sequence, since proteases break peptide bonds adjacent to preferred amino acid residues. In this exercise, students use peptide mapping to compare the structural relatedness of serum albumin from the human, cow, chicken and pigeon. Typical results of the exercise are shown below.
Peptide Mapping Analysis. Serum Albumin from human (lanes 2-4), cow (lanes 6-8), chicken (lanes 10-12), and pigeon (lanes 14-16) were digested with chymotrypsin prior to this separation by electrophoresis. Standard proteins are in lanes 1, 5, 9, and 13. 205. Protein Evolution and the Western Blot 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. Guided by step-by-step instructions, they subject serum proteins from six species to gel electrophoresis and transfer the separated proteins to nitrocellulose membranes. They then use an enzyme-linked immunoassay to compare the extent to which the separated albumins in the serum samples are related to those from cow. This exercise provides an exciting lesson in molecular evolution and introduces your students to one of the most important techniques of molecular biology. This exercise requires about two 2-3 hour laboratory sessions. 206. Affinity Chromatography 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. This exercise requires approximately two 2-3 hour laboratory periods and the materials provided to perform the experiment are shown below.
Price List - Standard Laboratory Program 2
Individual Experiments Each of the individual experiments is supplied with the chemicals and laboratory guides needed for 16 students working in pairs. (Please see page 17 for additional details). If you chose one or more of the experiments below, you should also order Electrophoresis Package 2M or Electrophoresis Package 2. Electrophoresis Package 2M provides sufficient agarose, gel stain, and electrophoresis buffers for 1 of the individual experiments in this series (four gels with 15ml of agarose per gel). Electrophoresis Package 2 provides sufficient agarose, gel stain, and electrophoresis buffers for up to 6 of the individual experiments in this series.
The following experiments include chemicals and instruction guides
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