Cell Fractionation and Centrifugation

Flashcards covering vocabulary, formulas, and stages of cell fractionation and centrifugation based on lecture notes.

Cell Fractionation

A laboratory technique used to break open cells and isolate specific cell components (organelles) while preserving their individual biological functions.

Homogenization

The stage of cell fractionation where cells are physically disrupted using a homogenizer or blender to release internal contents.

Homogenate

The liquid mixture created after cells have been physically disrupted to release their internal contents.

Mechanical Shearing

A physical method of grinding whole tissue using tools such as a mortar & pestle, blender, Dounce homogenizers, or Teflon homogenization.

Sonication

The use of high-frequency sound waves to shatter cell membranes during homogenization.

Osmotic Shock

A method of swelling and exploding cells via the use of a hypo-osmotic buffer.

French Press

A high-pressure method used to force cells through a narrow valve to disrupt them.

Filtration

A critical intermediate step using mesh or cheesecloth to remove large, un-ruptured intact cells and heavy fragments of connective cell walls from the raw homogenate.

Autolysis

The self-digestion of organelles, which is prevented during centrifugation by keeping the conditions cold to decrease enzyme activity.

Isotonic Condition

A pretreatment condition that maintains the same water potential as the cell to prevent osmotic bursting or shrinking.

Buffered Condition

A pretreatment condition that maintains a constant PH (usually $7.4$) to avoid denaturing cells.

Differential Centrifugation

A method where a mixture is spun at increasingly higher speeds to separate components based on sedimentation velocity.

Isopycnic Centrifugation

Also known as density or sedimentation equilibrium centrifugation, where particles separate solely based on buoyant density independent of time.

Sedimentation Coefficient ($S$)

A coefficient given by the formula S = \frac{1}{\text{\omega}^2 r} \times \frac{dr}{dt}, measured in Svedberg units.

Angular Velocity ($\omega$)

The rotor speed in radians/sec, calculated as $0.10472 \times \text{RPM}$.

Pellet

The larger and denser components of a homogenate that settle at the bottom of a tube during centrifugation.

Supernatant

The remaining liquid above the pellet after centrifugation, which contains smaller, less dense components.

Low-Speed Centrifugation

Centrifugation at $1,000 \times \text{gravity}$ for $10\,\text{minutes}$, typically resulting in a pellet containing whole cells, nuclei, and cytoskeletons.

High-Speed Centrifugation

Centrifugation at $80,000 \times \text{gravity}$ for $1\,\text{hour}$, resulting in a pellet containing microsomes and small vesicles.

Relative Centrifugal Force (RCF)

The force calculated when converting RPM using the formula $RCF = 1.118 \times 10^{-5} (r)(\text{RPM})^2$.

Density Gradient Centrifugation

A technique using a medium like sucrose or Percoll to create a gradient where particles migrate until they reach a region where their density matches the surrounding medium.

Marker Enzymes

Biochemical indicators used to determine the presence of specific organelles and quantify purified fractions.

Bradford Assay

A biochemical method using a spectrophotometer to determine the protein concentration of a fraction.

Specific Activity

The ratio of the activity of the enzyme of interest to the total protein concentration in a fraction.