BTEC-3200 Lectures 10 and 11 Practice Questions

Vocabulary practice flashcards covering biochemical separation techniques, Hydrophobic Interaction Chromatography (HIC), intracellular GFP isolation, and SDS-PAGE mechanics and reagents from BTEC-3200 Lectures 10 and 11.

Biofluorescence

A phenomenon in which organism proteins absorb UV light and re-emit it as visible light.

Bioluminescence

Light production that stems from a chemical reaction leading to light emission, differing from biofluorescence which involves UV light absorption by organism proteins.

Chromatography Matrix

The stationary phase medium in chromatography, which can be composed of materials such as paper, silica, hydrogel, or sand.

Hydrophobic Interaction Chromatography (HIC)

A separation technique based on reversible interactions occurring between hydrophobic regions of a target protein and hydrophobic ligands on the chromatography matrix.

High Ionic Strength Buffers (in HIC)

Buffers that shield electrostatic repulsion, enhancing stronger and more stable protein binding to the hydrophobic ligand of the HIC matrix.

Washing Phase (in HIC)

The phase in hydrophobic interaction chromatography that selectively removes contaminants possessing weak hydrophobic interactions.

Elution Phase (in HIC)

The phase that releases the target protein from the chromatography matrix, achieved by utilizing a buffer with a lower salt concentration.

Lysozyme (in GFP Isolation)

An enzyme involved in breaking down the bacterial cell wall during intracellular Green Fluorescent Protein (GFP) isolation.

Freeze/Thaw Cycles (in GFP Isolation)

A physical lysis method where ice crystal formation disrupts the cell wall and cell membranes to release intracellular GFP.

GFP Inherent Hydrophobic Nature

The intrinsic physical property of Green Fluorescent Protein that is directly exploited for its effective isolation and purification in HIC.

SDS-PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis)

An analytical electrophoresis technique whose primary purpose is to separate proteins strictly by molecular weight through a polyacrylamide gel matrix.

Sodium Dodecyl Sulfate (SDS)

A detergent that binds to non-polar regions of proteins, denaturing them and imparting a uniform negative charge so migration depends solely on molecular size.

Stacking Gel (in PAGE)

The upper portion of the gel cast with larger pores to align protein samples into tight, concentrated bands before entering the separating gel.

Bisacrylamide

A cross-linking agent that forms a cross-linked polymer network with acrylamide, stabilizing the gel matrix and preventing gel breakage and deformation during electrophoresis.

Tris-HCl Buffer (pH 6.8)

The buffer used in the stacking gel that maintains proteins in a partially protonated state, reducing their net negative charge and slowing migration to facilitate stacking into tight bands.

Separating Gel (in PAGE)

The lower, denser portion of the gel characterized by a higher acrylamide concentration and smaller pore sizes to resolve proteins based on molecular weight.

Acrylamide Functional Groups

The vinyl group ($-\text{CH}=\text{CH}_2$) and amide group ($-\text{C(O)NH}_2$) on the acrylamide monomer that enable polymerization into a gel matrix.

Ammonium Persulfate (APS)

A polymerization initiator that generates sulfate free radicals to start the polymerization of acrylamide monomers.

TEMED

A catalyst that accelerates acrylamide polymerization by catalyzing the decomposition of persulfate ions to generate free radicals.

Tris-HCl Buffer (pH 8.8)

The alkaline buffer in the separating gel that increases $\text{OH}^-$ ions, breaking protein ionic bonds to denature and linearize proteins for migration based strictly on size.

Glycine (in Running Buffer)

A trailing ion with a net negative charge that moves behind migrating proteins in SDS-PAGE, reducing friction and enhancing band resolution.

Coomassie Brilliant Blue

A staining dye that visualizes protein bands by binding via ionic and hydrophobic interactions to basic amino acids and aromatic residues, sensitive down to the microgram range.

Destaining Solution (SDS-PAGE)

A mixture typically composed of $20\%\text{ to }50\%$ methanol, $5\%\text{ to }10\%$ acetic acid, and water, formulated to remove excess background dye while leaving protein bands stained.

Methanol (in Destaining Solution)

The solvent component of destaining solution responsible for dissolving and washing away excess, unbound Coomassie Brilliant Blue.

Acetic Acid (in Destaining Solution)

The component of destaining solution that chemically fixes proteins in the polyacrylamide gel matrix to prevent them from diffusing or washing out.

Protein Band Intensity

A qualitative metric in gel visualization where a darker protein band indicates a higher concentration of that protein.

GFP Molecular Weight

The approximate molecular weight of Green Fluorescent Protein, which is $26\text{ to }28\,\text{kDa}$.