Protein Purification Techniques

• Seminar on protein purification

Selecting Starting Material

• The initial step involves choosing the right source material.

Purification Strategy

• A purification strategy guides the protein isolation process.

Classic Techniques for Protein Purification

• Common methods include centrifugation, chromatography, and electrophoresis.

Protein Specific Properties

• The charge, molecular size, solubility, and binding affinity for biological molecules allow the protein to be isolated.

Extraction and Homogenization

• The initial step is to break open cells to release their contents in a process called Homogenization.
• This can include cell localization, detergent use, and differential centrifugation.

Factors Influencing Purifications

• pH, ionic strength, temperature, and protease inhibitors are important.

Differential Centrifugation

• Differential centrifugation separates organelles before protein purification.
• Separates an organelle as a step prior to the purification of a protein

Centrifugation speeds and resulting fractions in the Isolation of Homogenate Components

• 500 x g for 10 minutes yields a nuclear fraction pellet.
• 10,000 x g for 20 minutes yields a mitochondrial fraction pellet.
• 100,000 x g for 1 hour yields a microsomal fraction pellet and the remaining soluble proteins in Cytosol.

Concentration

• Homogenization increases volume and decreases protein concentration.

Solubility's properties

• Used to change solubility of the protein across conditions

Concentration Methods

• Proteins can be precipitated using salts like ammonium sulfate.
• Changes in pH or temperature also cause precipitation.
• Organic solvents like ethanol and acetone are also utilized for the extraction of proteins.

Dialysis

• Used to reduce salt concentration in a solution.

Chromatographic Techniques for Protein Separation

• Chromatography separates by size (gel filtration), charge (ion exchange), ligand binding (affinity), and hydrophobicity.

Gel Filtration Chromatography

• Proteins are separated bases of size, via a gel filtration technique with exclusion limits.
• Uses Sephadex (dextran polymer) carbohydrate beads to separate molecules
• Larger molecules cannot enter the beads and are excluded.
• Small molecules can enter the aqueous area of the beads.
• Is in function of size (inside exclusion limits)

Ion Exchange Chromatography

• Used to separates compounds based on charge.
• A positively charged protein will bind to negatively charged beads.
• Conversely, negatively charged proteins flow through
• Carboxymethyl (CM) groups are examples as ionized forms.
• Diethylaminoethyl (DEAE) groups are example of protonated forms.

Affinity Chromatography

• Proteins are separated based on specifics
• A specific chromatography can be used for distinct proteins
• Glucose-binding proteins attach to glucose residues fixed on beads.
• When glucose is added, the glucose-binding proteins are released.

Methods to Detect and Quantify Proteins During Purification

• Protein concentration assessed via absorbance at 280nm, Biuret, or Bradford assays.
• Activity assays measure enzyme activity or function (transport, hormones).
• Other methods include electrophoresis, isoelectric focusing, and antibody use.

Absorbance

• Used to calculate absorbance in extinction coefficient

Defining Enzyme Activity

• Enzyme Activity measures the rate of a reaction (micromoles of product per minute).

Defining Specific Enzyme Activity

• Specific Activity indicates the number of enzyme units per milligram of total protein.

Polyacrylamide Gel Electrophoresis (PAGE)

• Electrophoresis on poliacrylamide gel determines sample purity.
• Determines properties like isoelectric point (pI), molecular weight, and number of subunits.

Electrophoresis Conditions

• Denaturing (PAGE-SDS) separates based on molecular weight.
• Non-denaturing conditions separate based on load and molecular weight.

Denaturing Agents

• Sodium dodecyl sulfate (SDS) and β-Mercaptoethanol are used.
• Proteins with subunits linked by disulfide bonds are denatured by heating with SDS and mercaptoethanol.
• Electrophoresis is then performed on the polyacrylamide gel.

Gel Formation

• The gel is a three-dimensional mesh of cross-linked and activated monomers.
• For polymerization, APS and TEMED act as catalysts which results in formation of radicals.

Determining Molecular Weight

• Protein mobility is conversely proportional to the logarithm of its molecular weight.

Western Blot Technique

• Proteins are transferred to a membrane, incubated with a specific antibody, and visualized via chemiluminescence, fluorescence, or radioactivity.

Antiboides

• Basic test for protein study

Isoelectric Focusing

• Separates based on relative amino acid content
• Protein separation occurs based on the creation of a pH gradient and application of an electric field
• Allows the proteins to be in the form of pl

2D Electrophoresis

• Proteins are separated by isoelectric point in the first dimension, followed by SDS-PAGE based on molecular weight.
• Separation happens based on the pl

Protein Identification Methods

• Methods include X-ray crystallography, MALDI-TOF mass spectrometry, measuring seqüència protèica, and Edman degradation.
• Protein crystallization is often necessary for X-ray crystallography.

X-Ray Crystallography

• Method used to visualize proteins

Mass Spectrometry (MALDI-TOF)

• Used to measuring the mass of a protein

MS-MS coupling

• Used to mesuring the mass and sequence of a protein

Protein Sequencing

• Large proteins are sequenced from smaller fragments
• Specific polypeptides are required for cleavage