Protein Isolation and Analysis Techniques

Flashcards covering cell fractionation, various chromatography methods, protein purification techniques (IP/Co-IP), SDS-PAGE, antibody types, Western blotting, and ELISA methods.

Cell fractionation

A technique designed to isolate and purify specific cellular organelles or structures in large quantities so their individual functions, proteins, and biochemical properties can be studied in isolation.

Homogenization

The first step of cell fractionation where cells are gently broken up in a blender or homogenizer to create a lysate or homogenate, rupturing membranes while keeping organelles intact.

Differential Centrifugation

A process that separates cellular components based on size and mass using a step-by-step increase in centrifugal speed.

Supernatant

The remaining liquid on top in a centrifuge tube after a spin that contains the smaller and lighter components.

Microsomes

Fragments of EB and go19; that are pulled into a pellet during the higher speed stage of differential centrifugation.

Gel-filtration Chromatography

Also known as size exclusion chromatography, it separates proteins based on their physical dimensions and is used to estimate molecular size.

Ion-Exchange Chromatography

A method that separates proteins based on their surface charge properties using a column packed with fixed-charge beads.

Cation exchange

A type of chromatography where beads are negatively (-) charged to trap positive proteins (cations).

Anion exchange

A type of chromatography where beads are positively (+) charged to trap negative proteins (anions).

Affinity Chromatography

The highest-resolution isolation method used to isolate one specific target protein from a mixture in a single step using beads covalently attached to a specific ligand.

The matrix (resin)

The solid, porous support structure of an affinity resin, usually made of agarose or polyacrylamide, that serves as the physical backbone.

Ligand

Highly specific molecules (such as a substrate, metal ion, or antibody) that biologically recognize and bind only to a target protein in affinity chromatography.

Spacer Arm

A small flexible chemical chain used to link a ligand to a matrix bead, providing physical clearance so the target protein can reach the ligand without being blocked by the bead.

Immunoprecipitation (IP)

A technique used to isolate and purify a single specific protein from a biological sample using a highly specific antibody and magnetic or agarose beads.

Co-immunoprecipitation (Co-IP)

A technique used to discover or confirm physical protein-protein interactions by pulling down a target protein along with any 'buddy' proteins physically bound to it.

SDS-PAGE

A technique used to separate a mixture of proteins strictly by their molecular weight to analyze purity or estimate size while eliminating variables of shape and charge.

SDS

An ionic detergent that denatures proteins into linear chains and wraps them in a uniform negative charge proportional to their mass.

Reducing Agent

Chemicals such as DTT or $\beta$-Mc used to break covalent disulfide bonds to ensure a protein is completely unraveled into single polypeptide strands.

Polyclonal Antibody

A heterogeneous mixture of different antibodies produced by many different B-lineages that recognize and bind to multiple different epitopes on the same antigen.

Monoclonal Antibody

A homogeneous collection of identical antibodies produced by a single, specific clone of B-cell that recognize one single, identical epitope with extreme precision.

Hybridoma technology

A laboratory method used to create monoclonal antibodies by fusing a single antibody-producing B-cell with an immortal cancer cell (myeloma).

Western blotting

A procedure used to detect, identify, and quantify a specific target protein within a complex mixture by separating proteins via SDS-PAGE and transferring them to a membrane.

Blocking

The step in western blotting where a membrane is soaked in a generic protein solution to coat empty spaces and prevent detection antibodies from sticking non-specifically.

Chemiluminescence

The emission of light produced when an enzyme (linked to a secondary antibody) reacts with a substrate, used to reveal the location of a target protein band.

ELISA

A highly sensitive diagnostic tool used to detect and quantify the presence of a specific substance (antigen or antibody) within a liquid sample using a 96-well microiter plate.

Indirect ELISA

A method primarily used to detect antibodies in a sample where the target antigen is coated onto the plastic surface of the well.

Sandwich ELISA

A method used to detect antigens in a sample by using a capture antibody and a detection antibody to trap the antigen between them.