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Isolation and Spectral Characterization of Chlorophyll-Protein Complexes

Abstract:: In this lab, groups of students will isolate chloroplasts membranes from spinach using centrifugation. After chlorophyll assay, thylakoid membranes are treated with a detergent concentration that results in the solubilization of large macromolecular complexes but not in complete membrane dissolution. Solubilized membranes are subjected to polyacrylamide gel electrophoresis to separate the green complexes. The green bands are sliced from the gel and their absorbance spectra measured, demonstrating the spectral differences between Photosystems 1 and 2. Students can use this technique to investigate differences due to growth conditions and plant species.

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Isolation of Myosin and Actin from Chicken Muscle

Abstract:: Using modifications of classic biochemical procedures for purifying the muscle contractile proteins, myosin and actin, we devised a 3-hour laboratory on the biochemistry of muscle that is appropriate for students in an introductory college biology course. The protocol begins with the extraction of small pieces of frozen chicken muscle in high salt (enhances myosin recovery) and low salt (enhances actin recovery) buffers. Solubilized myosin is induced to form visible filaments by lowering the ionic strength of the buffer, and a small pellet of enriched myosin is collected by centrifugation. Aliquots of both myosin and actin preparations are prepared for electrophoresis on small SDS, polyacrylamide slab gels. Completed electropherograms are stained, and students determine the molecular weights of gel bands. These data are then compared to known structural models of myosin and actin subunits and fragments.