Lecture 9: Cell Fractionation

Flashcards about Cell Fractionation.

Cell Fractionation

A technique to study cell structure and function by separating cell components and identifying their functions.

Cell Fractionation Purpose: Protein Enrichment

Concentrating proteins of a particular type in a biological sample for further analysis and identification.

Cell Fractionation Purpose: Protein Characterization

Identifying the subcellular localization, structure, and function of a protein.

Cell Fractionation Purpose: Protein Translocation

Monitoring how proteins move between cellular compartments, such as from the cytoplasm to the nucleus.

Homogenization

The process of breaking up tissue by mechanical force or chemical means to release organelles.

Lysis

The process where the inner contents of a cell leak out into the environment.

Homogenate

The solution containing organelles that leak out from cells after homogenization.

Centrifugation

A technique of separating substances by spinning samples at different speeds to isolate cell organelles/molecules.

Pellet

The sediment formed at the bottom of a centrifuge tube after centrifugation, consisting of particles with higher density than the solvent.

Supernatant

The liquid above the pellet in a centrifuge tube, containing particles that are lighter than the solvent and float to the top.

Low Speed Centrifuge

A centrifuge commonly used in labs, operating at room temperature with a maximum speed of 4000-5000 rpm.

Ultracentrifuge

An advanced centrifuge that separates smaller molecules at high speeds (up to 150,000 rpm) in a refrigerated, low-pressure chamber.

Differential Centrifugation

A procedure to separate organelles from whole cells for further analysis, based on size and density using repeated centrifugation at progressively higher speeds.

Velocity Sedimentation

A separation technique where a homogenate is layered on top of a density gradient solution (e.g., sucrose), and components separate into distinct bands during centrifugation.

Equilibrium Sedimentation

A separation technique where cell components are separated based on their buoyant density, independently of size and shape, using a steep density gradient (e.g., sucrose or caesium chloride).

Partitioning

A separation technique that isolates membrane vesicles based on differences in surface properties like charge and hydrophobicity.

Vacuole Function in Plants

Large internal aqueous space bounded by the tonoplast; maintains turgor pressure and stores nutrients and waste products.

Plant Cell Microbodies

Small vesicles in plant cells containing enzymes for oxidation of specific substrates, such as glycolate oxidation in peroxisomes.

Cationic Bead Method

A method to isolate plasma membranes from eukaryotic cells using positively charged beads to bind negatively charged cell surfaces.

Protoplasts

Plant cells that have had their cell walls enzymatically removed.

Cell Lysis Methods

Techniques that include sonication, enzymatic digestion, or high-pressure to disrupt the cell membrane.

Solubilization

The process of making a protein or molecule soluble by disrupting the cell membrane to release proteins.

Salting In

The process where the solubility of a protein increases at low salt concentrations due to the salt's ions masking charges on the protein.

Salting Out

A method of precipitating

Salting Out

A method of precipitating proteins from a complex mixture by adding high concentrations of salt.

Selective Precipitation

The process of selectively extracting proteins by adding specific salts or organic solvents that preferentially dissolve certain proteins.

Gel Electrophoresis

A visual method for monitoring protein purification steps by separating proteins based on charge and size.

Serum Protein Electrophoresis

An electrophoretic technique used to separate serum proteins into distinct bands, such as albumin, globulins (alpha, beta, gamma).

Isoelectric Focusing

A type of electrophoresis where proteins are separated based on their isoelectric point (pI) using a pH gradient.

2D Gel Electrophoresis

A two-dimensional electrophoresis technique that combines isoelectric focusing (IEF) and SDS-PAGE to separate proteins with high resolution.

IEF in 2D Electrophoresis

In 2D electrophoresis, the first dimension where proteins are separated based on their isoelectric point.

SDS-PAGE in 2D Electrophoresis

In 2D electrophoresis, the second dimension where proteins are separated based on their molecular weight.

Affinity Chromatography

Separates proteins based on the affinity of the protein for a specific ligand; the protein is specifically and reversibly bound to a matrix-bound ligand.

Size Exclusion Chromatography

A technique used to separate proteins based on their size and shape using a porous matrix.

What are the common steps in protein purification?

1. Cell lysis: Disrupting cells to release proteins. 2. Differential Centrifugation: Separating crude mixtures of cell homogenates using centrifugation. 3. Salt Fractionation: Separating proteins based on solubility. 4. Column Chromatography: Further separating proteins based on properties like size and charge.

What is Column Chromatography?

A purification method where a protein mixture is passed through a column containing a solid matrix, allowing separation based on properties like size, charge, or affinity.

What is the purpose of adding protease inhibitors during cell lysis?

To prevent the degradation of proteins by proteases released during cell lysis, ensuring the integrity of the target proteins.

Role of Ammonium Sulfate in Protein Precipitation

Ammonium sulfate is used to selectively precipitate proteins based on their solubility. By increasing the salt concentration, proteins with lower solubility will precipitate out of the solution, leaving other proteins behind.

Explain the principle of Ion Exchange Chromatography

Ion exchange chromatography separates proteins based on their net charge. The column is packed with charged beads, and proteins with the opposite charge bind to the beads, while others flow through. Bound proteins are then eluted by changing the salt concentration or pH.

Types of size exclusion chromatography

Gel-Filtration Chromatography: Separates particles by size, where smaller particles get trapped inside the pores of the column matrix and larger particles elute faster.

Describe the process of protein elution in affinity chromatography

In affinity chromatography, after the target protein binds to the specific ligand on the column, it is eluted by adding a high concentration of the ligand in the buffer, which competes with the matrix-bound ligand, releasing the protein. Alternatively, changing the pH or ionic strength can disrupt the binding.

How are proteins detected after electrophoresis?

Proteins are detected after electrophoresis using staining techniques like Coomassie blue or silver staining, which bind to proteins and make them visible. Alternatively, if the proteins are labeled (e.g., with fluorescent tags), they can be detected using fluorescence imaging.

Purpose of SDS-PAGE

SDS-PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis) is