Experiment 1

Experiment 1 - BC368 Biochemistry of the Cell II Experiment...

Info iconThis preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon
1 BC368 – Biochemistry of the Cell II Experiment 1 Characterization of the Plasma Membrane Introduction Biological membranes separate the inner workings of cells from the outside environment. Moreover, eukaryotic cells are divided into compartments, with the interiors of different organelles separated from each other and from the cytoplasm by membranes. Animal membranes are composed primarily of lipids, cholesterol, and proteins arranged in a fluid bilayer. In this two-week exercise you will characterize the components and properties of the red blood cell plasma membrane. Red blood cells are commonly used in membrane investigations because they contain only the plasma membrane (in most animals). The specific goals of this work are as follows: identification of the protein components of the plasma membranes of sheep erythrocytes. characterization of the protein distribution across the two halves of the plasma membrane. development of a model of membrane permeability using artificial membranes. Protein Components of the Plasma Membrane Membranes have the potential for the components of the two leaflets to be distributed asymmetrically. In this part of the exercise we will attempt to characterize the protein components of the red blood cell plasma membrane in terms of their distributions across the membrane. When the fluorescent dye fluorescein isothiocyanate (FITC) is added to intact cells, it binds to free amino groups of proteins exposed on the outside of the cells, thereby labeling them. Alternatively, if membrane fragments, instead of whole, intact cells, are prepared and exposed to FITC, membrane proteins that are exposed on either side of the membrane will be labeled by the FITC. After FITC-labeling proteins in both samples, the membranes can be disrupted and the proteins contained within them separated by polyacrylamide gel electrophoresis. Those proteins that are fluorescently labeled can be visualized as orange bands under UV light (302 nm). You can then use the molecular weights of proteins labeled in the whole cells versus those in the membrane fragments to identify proteins exposed only on the outside surface of cells, those exposed only on the inside surface, and those that extend across the membrane. Membrane Permeability When amphiphilic molecules are placed in aqueous solution, they spontaneously arrange themselves into structures called micelles . In these structures the polar head groups are on the outside, interacting with water, and the nonpolar tails are on the inside, interacting with each other. This arrangement serves the dual purpose of minimizing unfavorable contacts between water and the hydrophobic tails of the amiphiphiles while maximizing solvation of the polar head O HO O COOH N C S FITC
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
BC 368- Experiment 1- Spring 2010 2 groups. A suspension of phospholipids in water results in the spontaneous formation of liposomes : closed, self-sealing, solvent-filled vesicles bounded by a single bilayer. Such liposomes provide good experimental models for biological membranes. One crucial function of biological membranes is to isolate and protect the numerous
Background image of page 2
Image of page 3
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 02/09/2010 for the course CH CH242 taught by Professor Katz during the Spring '10 term at Colby.

Page1 / 7

Experiment 1 - BC368 Biochemistry of the Cell II Experiment...

This preview shows document pages 1 - 3. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online