BioChem Section 4.1 Purification of Proteins

Flashcards for Protein Purification Lecture Notes

Protein Purification

Proteins can be purified based on physical properties like size and charge.

Assay in Protein Purification

An assay is required to detect the presence of the protein of interest during purification.

Assay for Lactate Dehydrogenase

The assay detects enzyme activity by monitoring NADH spectrophotometrically

Specific Activity ( Analyzing a Purification Scheme )

Enzyme activity / Total protein

Goal ( Analyzing a Purification Scheme)

Maximize specific activity.

Step 1: ( Released from the Cell to be purified)

Disrupt cell membranes to create a homogenate.

Step 2: ( Released from the Cell to be purified)

Centrifuge homogenate at low speed to yield. Pellet consisting of heavy material and lighter supernatant

Step 3 : ( Released from the Cell to be purified)

Centrifuge supernatant at higher centrifugal force yield another pellet and supernatant.

Differential Centrifugation

A process of repeated centrifugation of homogenate to yield fractions of decreasing density. It helps enrich the desired protein.

Salting Out

Proteins are less soluble at high salt concentrations, causing them to precipitate.

Dialysis

Separates proteins from small molecules using a semipermeable membrane, where Large proteins remain inside and small molecules diffuse out.

Gel-Filtration Chromatography

Separates proteins based on size using a column with porous beads; large proteins exit first, and small proteins exit last.

Ion-Exchange Chromatography

Separates proteins based on charge using a column with charged beads; elution is done by increasing salt concentration. Proteins with same charge flow through quickly. Proteins with opposite charge bind to beads.

Cation Exchange

Negatively charged beads that bind positive proteins.

Anion Exchange

Positively charged beads that bind negative proteins.

Affinity Chromatography

Uses beads with specific ligands attached; proteins with affinity for the ligand are retained and then eluted.

High-Performance Liquid Chromatography

Uses fine beads and pressure to force buffer through the column, resulting in sharper, faster separations.

Gel Electrophoresis

Separates proteins based on net charge using an electric field; Used to separate proteins and nucleic acids. Small proteins move faster through the gel.

SDS-Page

Detergent that denatures proteins and gives them a uniform negative charge, allowing proteins to migrate based on mass only (mass ratio). SDS binds about 1 molecule per 2 amino acids

Western Blotting Permits the Detection

Primary antibody is an antibody specific for the protein. Secondary antibody is an antibody specific for the primary antibody's shape.

Western Blotting Permits the Detection

Western Blotting is proteins are separated in an SDS-Page gel and then transferred to a polymer. Stained with a primary antibody, stained with a secondary antibody, and quantified.

Isoelectric Focusing

Separates proteins in a pH gradient gel based on their isoelectric point (pI). Where it has no net charge.

Two-Dimensional Electrophoresis

Two-dimensional electrophoresis separates proteins in two directions. First dimension: isoelectric focusing (horizontal) by pI. Second dimension: SDS-Page in a perpendicular (vertical) by mass.

Quantitative Evaluation

Measure purification effectiveness by tracking changes in total protein, total activity, specific activity, yield, and purification fold.

Ultracentrifugation

Separates molecules based on mass, density, shape, and interactions; used for analyzing protein structure and complexes.

Sedimentation Coefficients

Measure rate of sedimentation during centrifugation, expressed in Svedberg units (S); The smaller the S value, the more slowly a molecule moves. Elongated particles sediment more slowly than compact ones.

Recombinant DNA Technology

Enables large-scale protein expression, addition of affinity tags for purification or visualization, and primary structure modifications.