March 6

The presentation and accompanying slides are the exclusive copyright of Professor Omri.
Usage is restricted to enrolled students in Biochemistry I (CHMI-2227 E) at Laurentian University for Winter term 2026.
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Also known as: Size exclusion chromatography or molecular sieve chromatography.
Mechanism:
- Utilizes beads with specific-sized pores.
- Filter Action:
- Larger proteins cannot enter the pores and elute first due to less volume to traverse.
- Smaller proteins enter the pores, causing delayed elution and thus elute later.
Separation Basis:
- Proteins are separated based on molecular mass, with larger proteins eluting earlier and smaller proteins later.
Bead Variations:
- Various beads with different pore size ranges are available for tailored selection in separation processes.

Employs matrix, often dextran (Sephadex ®), which forms a mesh with cross-linking that restricts penetration to specific-sized molecules.
Alternative Matrices: Agarose and sepharose are alternative materials for creating matrices.
Elution Mechanism:
- Smaller molecules, having more time inside the beads, elute later compared to larger molecules.

Principle: Utilizes specific biological properties for protein purification.
Binding Mechanism:
- Proteins bind to a covalently attached ligand on the matrix within the column.
Elution Method: Achieved using a high salt concentration or a pH solution.
Ligands Used:
- Can be antibodies, substrates, metals, or other macromolecules that interact with the target protein.

When filtering a mixture of partially purified proteins, only the protein of interest binds to the ligand, while contaminants are washed away.
The protein of interest is eluted by adding an excess of unbound ligand.

Purpose: Separates protein molecules based on charge and size.
Mechanism:
- Proteins migrate through a gel matrix, which can be polyacrylamide or agarose, under the influence of an electric field.

Staining: After electrophoresis, gels are stained using dyes that bind to proteins or protein transfer to a nitrocellulose membrane via electroblotting.
Immunoblotting: Known as Western blotting, this involves probing the proteins with specific antibodies.

Usage: Employs SDS (sodium dodecyl sulfate), a negatively charged detergent, to denature proteins.
Mechanism:
- SDS disrupts the native protein conformation causing proteins to adopt a linear shape.
- Mercaptoethanol is added to cleave disulfide bonds.
Molecular Weight (MW) Estimation: Achieved using standard proteins of known MW run on the same gel.

Illustrated by a tetrameric protein with peptides connected by disulfide bonds.
Cleaves into monomeric proteins and separated peptides after heating with SDS and mercaptoethanol.

Historical Development: Developed by Swedish scientist T. Svedberg in 1923.
Process: Uses an ultracentrifuge that can generate centrifugal forces exceeding 600,000 g.
Applications:
- Determining molecular weight and subunit composition of proteins.
- The sedimentation rate of proteins is influenced by their shape and molecular weight.

Fix an antibody on a solid support, typically a 96-well polystyrene plate.
Incubate the protein sample; the desired protein will bind to the antibody.
Wash away unbound or unwanted proteins.
Add a second antibody linked to an enzyme.
The enzyme catalyzes a reaction forming a colored product.
The intensity of the color is proportional to the concentration of the protein in the sample.

Method: Determines protein concentration by measuring absorbance at 280 nm.
Contributors: Aromatic amino acid residues (Tryptophan, Tyrosine, Phenylalanine) contribute to absorbance.
Key Features:
- Quick, sensitive, and non-destructive to protein samples.
- Linear relationship exists between protein concentration and absorbance.

Process: Treatment of a protein sample with alkaline CuSO4 forms a purple complex with copper ions and nitrogen atoms of peptide bonds.
Detection:
- Absorbance is monitored at 540 nm.
- This method is not highly sensitive and is destructive to samples.
- Susceptible to interference from ammonia or ammonium ions.

Variation: Similar to the Biuret method but incorporates an additional reagent for enhancing color development.
Reagent: Folin-Ciocalteau is a combination of phosphomolybdate and phosphotungstate salts which reacts with phenols (such as tyrosine side chains).
Color Measurement: Absorption measured at 600-750 nm.
Sensitivity: Approximately 100 times greater than the Biuret method, but requires more time to perform.

Mechanism: Utilizes Coomassie brilliant blue G-250 dye that binds proteins in acidic solutions.
Detection: Color change monitored at 595 nm.
Advantages: Simple and highly sensitive method.

Variation: A modification of the Lowry method where BCA replaces Folin-Ciocalteau reagent.
Chemical Reaction: BCA reacts with Cu2+; in an alkaline medium, forms a purple complex with Cu2+.
Absorbance Measurement: Typically measured at 562 nm.
Sensitivity: Comparable to the Lowry method, destructive to protein samples.

Essential for understanding the amino acid sequence and primary structure of proteins.
Crucial for determining three-dimensional structure, molecular mechanisms of action, and developing therapies for inherited diseases.
Enables the development of diagnostic tests for inherited diseases based on amino acid changes.

Separation: If multiple polypeptide chains are present, they must be separated and purified.
Disulfide Bridge Reduction: Cleaving (reducing) disulfide bridges occurs.
Terminal Residues: Determine N- and C-terminal residues.
Fragmentation: Cleave each chain into smaller fragments and determine the sequence of each fragment.
Repetition: Repeat fragmentation using different cleavage procedures to generate overlapping fragments.
Reconstruction: Reconstruct the protein's sequence from the overlapping fragment sequences.
Disulfide Cross-link Identification: Determine the locations of any disulfide cross-links.