Cell fractionation and Ultracentrifugation

Cell Fractionation

• A method used to study the structure and functions of various component of a cell - organelles

• It enables an efficent and cheap isolation of the biological material in larger quantities

• This involves breaking up the cells and separation of different organelles (cellular components)

Properties of a solution used in cell fractionation

• Ice cold - Limits enzyme activity, especially hydrolytic enzymes in lysosomes

• pH buffered - prevents denaturing of proteins, maintaining protein structure in the cell membrane

• Isotonic - prevents osmotic lysis

Stage 1 - homogenation

• Tissue is typically homogenized by the means of grinding, mincing, chopping, pressure changes, osmotic shock, freeze-thawing, and ultra sound.

• The resultant fluid is known as homogenate, and its additionally filtered to separate from any remaining unbroken cells and larger debris

Stage 2 - ultracentrifugation

• The centrifuge is used to create centrifugal force; the homogenate spun at increasingly higher speeds is exposed to greater centrifugal force

• The most dense organelles separate out first from the homogenate by forming a pellet at the bottom of the tube

• The remaining liquid part is the supernatant

• Its then spun again at a greater speed and time

• This results in subsequently smaller structures becoming isolated