EVERGREEN INS 2008 - Cell Fractionation by Differential Centrifugation

Unformatted text preview:

Cell Fractionation by Differential Centrifugation 2007-08 INS Winter Quarter – Lab 2 The purification of a specific macromolecule often begins with the isolation of a particular cellular organelle. For example, if we were interested in studying the organization of chromatin in yeast cells, we could start by isolating nuclei. In a cell fractionation procedure, the cell must first be disrupted by breaching the cell boundaries. Common methods include grinding in a mortar, homogenization in ground glass, and exposure to sonic vibrations. In most cases, disruption is carried out at low temperatures (between 0o and 4oC) to minimize proteolysis and loss of biological activity. After cell disruption, the particulate components are separated from each other by high speed centrifugation. In differential centrifugation, the heavier particles settle first, followed by gradual separation of lighter particles by sedimentation. For example, centrifugation at a speed 600 times gravity of a tissue homogenate suspended in 0.25 M sucrose results in the separation of nuclei. If the supernatant resulting from this centrifugation is centrifuged at 10,000 times gravity, the mitochondria are pelleted out of solution. Centrifugation at still higher speeds (100,000 times gravity) pellets other particulate components, such as the microsomal fraction (endoplasmic reticulum). [The relative centrifugal force (RCF) is expressed by using g units, where 1 g is the force of the earth’s gravity. The force exerted on a particle in a centrifuge is expressed by the following equation: RCF = 1.119 x 10-5 (rpm2) r where rpm is the revolutions per minute of the rotor and r is the distance (in cm) of the particle from the axis of rotation.] Resolution of cellular structures under the light microscope can be increased by fixation and staining of tissue samples. Fixation involves the selective preservation of morphological organization and chemical content for microscopic observation. The most reliable fixatives contain one or more substances that precipitate the proteins of the cell or render them insoluble without precipitating them. Staining involves chemical reactions between a dye and proteins or nucleic acids. The selection of a specific stain depends on a number of factors including the nature of the material to be studied, the type and pH of the fixative used, and the chemical reactivity of the dye. We will be using two stains in our experiments today: aceto-orcein and Janus green. Aceto-orcein specifically stains nuclei. It contains a fixative, so both staining and fixing are achieved in one step. Janus Green B specifically stains mitochondria, and is an example of a substance known as a vital stain. When Janus Green is introduced into a cell, it stains all of the cell components green, since it is maintained in its oxidized state. After a brief time, however, it is reduced to its colorless form in all parts of the cell except the mitochondria, where it is reoxidized by cytochrome oxidase (an enzyme that takes part in the final stages of cellular respiration) and retains its color. Laboratory aims In the laboratory, you should generate fractions enriched for nuclei and mitochondria, starting from a cauliflower cell homogenate. After completing this lab, you should understand 1) the principles and methods of tissue homogenization and differentialcentrifugation; and 2) how organelles can be visualized through the use of specific chemical stains. Protocol Overall strategy This lab is aimed at isolating a nuclear fraction and mitochondrial fraction from cauliflower cells using the method of differential centrifugation. Cauliflower tissue will be homogenized in a mortar and pestle with a buffered, isotonic sucrose solution and a quantity of sand. The homogenate will first be filtered through cheesecloth to remove larger debris and then centrifuged at a relatively low speed to pellet whole cells and the nuclear fraction. The supernatant will then be centrifuged at a relatively high speed to pellet the mitochondrial fraction. The starting material and each fraction will be reacted with chemical stains that will allow better visualization of these organelles under the compound microscope.Reagents Homogenization buffer: 0.4 M sucrose, 50 mM potassium phosphate pH 7.7, 5 mM EGTA Resuspension buffer: 20 mM potassium phosphate, pH 7.0, 20 mM 2-mercaptoethanol Part A: Homogenization and cell fractionation Important: Work on ice for all steps! 1. Chill cauliflower heads (the heads should be firm and tight, not in the flowering or “loose” stage). Carefully use a razor blade to cut the outer 5-10 mm onto aluminum foil over ice. Begin with about 5 g of cauliflower. 2. Using a chilled mortar and pestle, grind the cauliflower with 2.5 g clean sand for 30 sec. Add 15 ml of homogenization buffer and grind an additional 30 sec to suspend the mixture. Pour off into a 50 ml plastic centrifuge tube. Rinse the mortar with 5 ml of buffer and combine it in the centrifuge tube with the original mixture. Let the sand settle to the bottom for 2 min on ice. 3. Filter this mixture through four layers (2 pieces) of cheesecloth into a chilled, 30 ml centrifuge tube. Using gloves, squeeze fluid from cheesecloth into the tube. This is your INITIAL HOMOGENATE. Save a 0.5 ml aliquot of the homogenate in a labelled Eppendorf tube. 4. Using a balance, match your tube with another group’s tube to within 0.1 g. Centrifuge at 600 xg for 10 min at 4oC. 5. Decant the supernatant into a clean, chilled 30 ml centrifuge tube. The pellet is your NUCLEAR PELLET. Keep the nuclear pellet on ice. 6. Match your group’s tube with another group’s tube to within 0.1 g. Centrifuge the post-nuclear supernatant at 10,000 xg for 15 min at 4oC. 7. Save 0.5 ml of the FINAL SUPERNATANT into a labelled Eppendorf tube (it should be fairly clear). Discard the remainder of the supernatant. The pellet is your MITOCHONDRIAL PELLET. Keep the mitochondrial pellet on ice. 8. At the end of this procedure, you should have four fractions: a) 0.5 ml of the INITIAL HOMOGENATE b) NUCLEAR PELLET c) MITOCHONDRIAL PELLET d) 0.5 ml of the FINAL SUPERNATANT Add 2.0 ml of the ice-cold resuspension buffer to the MITOCHONDRIAL PELLET. Scrape the pellet from the wall of the tube with a clean spatula and resuspend by vortexing. Part B: Characterization by microscopy Examination without staining


View Full Document

EVERGREEN INS 2008 - Cell Fractionation by Differential Centrifugation

Download Cell Fractionation by Differential Centrifugation
Our administrator received your request to download this document. We will send you the file to your email shortly.
Loading Unlocking...
Login

Join to view Cell Fractionation by Differential Centrifugation and access 3M+ class-specific study document.

or
We will never post anything without your permission.
Don't have an account?
Sign Up

Join to view Cell Fractionation by Differential Centrifugation 2 2 and access 3M+ class-specific study document.

or

By creating an account you agree to our Privacy Policy and Terms Of Use

Already a member?