LIFESCI 23L Lecture Notes - Lecture 6: Size-Exclusion Chromatography, Gel Electrophoresis, Agarose Gel Electrophoresis
Lecture F: Gel Electrophoresis
OVERVIEW OF SKILLS/CONCEPTS
● Skills: micropipetters, SDS-Page gel, creating standard curve in Excel, determining size of unknown
polypeptide using standard curve, loading/running agarose gel, use of spectophotometer to determine
DNA concentration
BACKGROUND
● Protein structure
○ Proteins: primary function is structure/metabolism; also invovled in catalysis, energy generation,
transport, signaling, recognition function, fibers (e.g. hair collagen muscle), and globular (e.g.
enzymes)
○ Polymers of amino acids; assembled on the carboxyl side, read from N-terminal → C-terminal
○ Basic amino acid=zwitterion → 20 amino acids that differ in side groups → grouped into different
groups (polar charged, polar uncharged, nonpolar hydrophobic)
■ Most fundamental protein structure =primary structure
● Primary: amino acid sequence ; determines secondary and tertiary structure
● Secondary: local interactions of amino acids and backbones (alpha helixes and
beta sheets)
○ Beta sheets can be parallel or antiparalle
○ Proteins fold depending on environment (hydrophilic or hydrophobic)
● Tertiary: overall shape of protein molecule; how units of secondary structure are
folded together
○ DISULFIDE BOND most important element of tertiary (and quarternary)
structure for this lab
○ Cysteine (an amino acid) residues from different parts of a protein, or
different polypeptide chains, can be covalently linked by disulfide bonds
■ Contribute greatly to 3D protein structure and stabilize protein
shape in different chemical environments
■ These covalent bonds frequently hold subunits together and help
proteins fold into final conformations
■ Disulfide linkages intramolecular)
● Quarternary structure: overall shape of 2 or more polypeptide chains (subunits)
together to form functional protein --disulfide linkages (intermolecular)
○ Homo: subunits identical; hetero: subunits different
○ dimer=2 units, trimer=3 units, etc.
● Protein chrystallography: uses protein crystal and x-rays to produce refraction patterns → e- density can
be calculated and protein structure can be modelled
○ To do so, one must purify a single protein from all other components of the cell
○ How?
○ Protein purification
■ Proteins of different ize can be separated via ultracentrifugation
● Proteins put in sugar gradient and centrifuged strongly; proteins travel until they
reach the density of gradient that matches their own; different fractions can then
be removed and analyzed
○ Column Chromatography
■ Depending of matrix in the column, proteins can be separated by size, hydrophobicity,
charge, or binding to specific groups
● Size exclusion chromatography (larger molecules elude first, then small)
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Skills: micropipetters, sds-page gel, creating standard curve in excel, determining size of unknown polypeptide using standard curve, loading/running agarose gel, use of spectophotometer to determine. Proteins: primary function is structure/metabolism; also invovled in catalysis, energy generation, transport, signaling, recognition function, fibers (e. g. hair collagen muscle), and globular (e. g. transport, signaling, recognition function, fibers (e. g. hair collagen muscle), and globular (e. g. enzymes) Polymers of amino acids; assembled on the carboxyl side, read from n-terminal c-terminal. Basic amino acid=zwitterion 20 amino acids that differ in side groups grouped into different groups (polar charged, polar uncharged, nonpolar hydrophobic) Primary: amino acid sequence ; determines secondary and tertiary structure. Secondary: local interactions of amino acids and backbones (alpha helixes and beta sheets) Beta sheets can be parallel or antiparalle. Proteins fold depending on environment (hydrophilic or hydrophobic) Tertiary: overall shape of protein molecule; how units of secondary structure are folded together. Disulfide bond most important element of tertiary (and quarternary) structure for this lab.
