BIOL201L Lab 5: Cell Fractionation and Biochemical Assays Adapted from Choinski,
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BIOL201L Lab 5: Cell Fractionation and Biochemical Assays Adapted from Choinski, Experimental Cell and Molecular Biology, 2nd ed, 1992 INTRODUCTION Eukaryotic cells are distinguished from prokaryotic cells by the presence of internal, membrane-bound structures that perform specific functions for the cell. The membranes serve the purposes of grouping together reactions that are similar and/or in similar pathways, as well as concentrating the reactants for the chemical reactions through membra transport. These internal organelles are part of the cell, and can be removed whole from the cell with intact functions. The process through which we can remove the organelles is called differential centrifugation, the process of grindingu whole cells to release the organelles and membranes, and then separating the different types of organelles according density through centrifugation. Centrifuges apply force on matter that is in a suspension, like organelles suspended in cytoplasm, so needed to force the matter to the bottom of the tube is inversely proportional to the density of the matter: heavier larger items can be pelleted at lower speeds, separate organel that the speed while lighter, smaller items must be pelleted at higher speeds. Thus, to lles of different sizes from cells, we can use different speeds to produce different fractions that correspond to heavy, medium, and light organelles. QUESTION 1:What organelles would you expect to be in a heavy fraction? a medium fraction? a light fraction? Cell fractionations are usually carried out in a buffer medium that adds density to the overall solution, such as 0.25 sucrose/ 25 mM potassium chloride. This allow further differentiate between organelles with similar densities. In this lab, we will use a very simple stepwise metho fractionation, since we do not need very pure fractions-we will simply "enrich" the fractions with the organelles want. This is accomplished by centrifuging the mixture at low speed, then separating the supernatant from the pell and re-centrifuging the supernatant. At the end of the process, each group will have a set of fractions (pellets and supernatants) that are enriched in specific organelles as shown in the flowchart below. s the buoyancy of the medium to counteract the organelle density ar sample cell homogenate centrifuge 10 min at 600Xg let supernatant, S1 centrifuge 10 minutes at 3000 xg P1: nuclear fraction pellet supernatant S2 resuspend centrifuge 15 minutes at 15,000 x S3Explanation / Answer
Heavy fraction will have organelles like nucleus. Besides nucleus, whole cells and cytoskeleton is also found in the heavy fraction. The medium fraction will have chloroplasts, mitochondria, lysosomes, and peroxisomes. The light fraction will have microsomes, other small vesicles, and ribosomes. Nucleus, cell membrane, cell wall, chloroplasts, chromoplasts, and vacuoles can be observed without staining. However, ribosomes, mitochondria, endoplasmic reticulum, golgi apparatus need to be either stained for observation or they can be observed under an electron microscope. Lysosomes can be isolated in the medium fractions. So, medium fractions would test positive for lysosomal enzyme activity. In the P1 fraction we are looking for nuclei. Some of the enzymes that can be found in this fraction are DNA polymerase, RNA polymerase, NAD synthetase, nucleotide phosphorylase etc. In the P2 fraction we are looking for organelles like mitochondria, chloroplasts, lysosomes, and peroxisomes. Some of the enzymes that can be found in this fraction include catalase, cytochrome oxidase, malate dehydrogenase, monoamine oxidase, uric acid oxidase etc. In the P3 fraction organelles like microsomes, ribosomes, and other small vesicles are present. Some of the enzymes that can be found in this fraction include cytochrome P450 enzymes, NADPH-cytochrome c reductase, peptidyl transferase etc. Catalase enzyme is usually found in peroxisomes. Peroxisomes are isolated in P2 fraction. So P2 fraction will have highest activity of catalase enzyme. The nuclear fractions or the P1 fractions have very low to nonexistent activity of catalase enzyme.
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