BIOL232 Protein Isolation (Extraction) and Quantification

Protein Function (Enzymes)

Thousands of them, such as Rubisco and alcohol dehydrogenase.

Protein Function (Structural)

Provides mechanical support, e.g., collagen.

Protein Function (Motor)

Generate movement, e.g., myosin and flagellin.

Protein Function (Receptor)

Detect signals, e.g., Rhodopsin in retina or insulin receptor.

Protein Function (Storage)

Stores amino acids or nutrients, e.g., Casein and endosperm.

Protein Function (Transport)

Carry ions or small molecules, e.g., Serum albumin.

Protein Function (Regulation)

Controls gene expression, e.g., DNA binding proteins and transcriptional activators.

Protein Function (Signaling)

Transmits signals, e.g., stress response hormones, growth factors, and EGF.

Protein Structure (Primary)

The unique linear sequence of amino acids held together by covalent peptide bonds.

Protein Structure (Secondary)

Local folding patterns like the alpha helix and beta sheet.

Protein Structure (Tertiary)

The three-dimensional conformation achieved by folding into the lowest energy state, determining function/properties.

Protein Structure (Quaternary)

The structure resulting from multiple polypeptide chains (subunits) associating together.

Protein Denaturation

A change in chemical structural and biological properties of proteins leading to loss of structure.

Denaturation Effect on Structure

Loss of secondary, tertiary, and quaternary structures, but the primary structure remains unaffected.

Causes of Denaturation

Extreme temperatures (heat or radiation), changes in pH, sonication/mechanical shearing, or chemicals (e.g., SDS, solvents, alcohols).

Protein Renaturation

The process where some denatured globular proteins can regain their activity and correct folding.

Good Buffers

Buffers that have low metal binding capabilities and are relatively free of side effects, e.g., Tris buffers and Phosphate buffers.

Thiol Compounds in Buffer

Added to protect proteins from oxidation, e.g., DTT or 2-mercaptoethanol (beta-ME).

Chelating Agents in Buffer

Protect enzymes from inactivation by heavy metal ions and prevent protein-metal ion aggregation/precipitation, e.g., EDTA.

Protease Inhibitors in Buffer

Added for suppression of endogenous proteases, e.g., PMSF.

Osmotically Active Solutes in Buffer

Maintains the tonicity of the solution comparable to the cell/tissue to avoid swelling or plasmolyzing of organelles.

Detergents in Buffer

Added to solubilize organelles or membrane associated proteins.

Example of a Non-ionic Detergent

Triton X-100, commonly added at 0.1-0.5%.

PVPP (Polyvinylpolypyrrolidone)

Added to plant extracts (2-10% w/w) to prevent 'browning' from alkaloids and polyphenolic compounds.

Protein Quantification Method (UV Absorbance)

Simple but highly error prone.

Protein Quantification Method (Biuret Assay)

Involves protein-copper chelation; compatible with most surfactants but incompatible with substances that reduce copper.

Protein Quantification Method (Calorimetric Dye-based)

Involves protein-dye binding and direct color change detection; incompatible with surfactants but compatible with most salts/buffers/reducing substances.

Protein Quantification Method (Bradford Assay)

A coomassie dye-binding calorimetric assay; detects protein concentration in the range of 2 to 1500 ug/mL

Bradford Assay Principle

In an acidic environment, proteins bind to coomassie dye, causing a spectral shift from reddish-brown to blue.

Bradford Assay Measurement Wavelength

595 nm, which is the optimal wavelength to measure the blue color of the Coomassie dye-protein complex.

Amino Acids detected by Bradford Assay

Basic amino acids: primarily arginine, lysine, and histidine.

Lab Protein Extraction Method

Lysis by mechanical grinding in extraction buffer, followed by centrifugation.

Total Protein Quantification Method (Lab)

Bradford assay using a standard curve.

Standard Curve purpose

Used to calculate the unknown concentration of protein extracts.

Final Calculation Step (Concentration)

Must account for the dilution factor to determine the total amount of proteins in the original extract.