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.
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.
In this experiment, students prepare a protein extract from cow heart. They then determine the molecular weights of major contractile proteins by comparing their migration on SDS-polyacrylamide gels to the migration of standard proteins of known size. Students also identify and determine the molecular weight of the major proteins found in milk. This exercise requires 1 three-hour or 2 two-hour lab sessions and a table top centrifuge or microcentrifuge is required. Typical results of the exercise are shown on the gel.
The five histone molecules are the major proteins that associate with DNA to form nucleosomes. In this experiment, students isolate nuclei from chicken erythrocytes and then prepare nucleosomes from the erythrocytes by using a simple procedure that employs micrococcal nuclease. They then determine the molecular weights of the five histone proteins by comparing their migration on SDS-polyacrylamide gels to the migration of standard proteins of known size. This exercise requires a single two-three hour lab session and a microcentrifuge is required.
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).