BIOCHEM C785 UPDATED ACTUAL EXAM
QUESTIONS AND CORRECT ANSWERS
●● FMOC Chemical Synthesis
Answer: Used in synthesis of a growing amino acid chain to a
polystyrene bead. FMOC is used as a protecting group on the N-
terminus.
●● Salting Out (Purification)
Answer: Changes soluble protein to solid precipitate. Protein precipitates
when the charges on the protein match the charges in the solution.
●● Size-Exclusion Chromatography
Answer: Separates sample based on size with smaller molecules eluting
later.
●● Ion-Exchange Chromatography
Answer: Separates sample based on charge. CM attracts +, DEAE
attracts -. May have repulsion effect on like charges. Salt or acid used to
remove stuck proteins.
●● Hydrophobic/Reverse Phase Chromatography
,Answer: Beads are coated with a carbon chain. Hydrophobic proteins
stick better. Elute with non-H-bonding solvent (acetonitrile).
●● Affinity Chromatography
Answer: Attach a ligand that binds a protein to a bead. Elute with harsh
chemicals or similar ligand.
●● SDS-PAGE
Answer: Uses SDS. Gel is made from cross-linked polyacrylamide.
Separates based off of mass with smaller molecules moving faster.
Visualized with Coomassie blue.
●● SDS
Answer: Sodium dodecyl sulfate. Unfolds proteins and gives them
uniform negative charge.
●● Isoelectric Focusing
Answer: Variation of gel electrophoresis where protein charge matters.
Involves electrodes and pH gradient. Protein stops at their pI when
neutral.
●● FDNB (1-fluoro-2,3-dinitrobenzene)
,Answer: FDNB reacts with the N-terminus of the protein to produce a
2,4-dinitrophenol derivative that labels the first residue. Can repeat
hydrolysis to determine sequential amino acids.
●● DTT (dithiothreitol)
Answer: Reduces disulfide bonds.
●● Iodoacetate
Answer: Adds carboxymethyl group on free -SH groups. Blocks
disulfide bonding.
●● Homologs
Answer: Shares 25% identity with another gene
●● Orthologs
Answer: Similar genes in different organisms
●● Paralogs
Answer: Similar "paired" genes in the same organism
●● Ramachandran Plot
Answer: Shows favorable phi-psi angle combinations. 3 main "wells"
for α-helices, ß-sheets, and left-handed α-helices.
, ●● Glycine Ramachandran Plot
Answer: Glycine can adopt more angles. (H's for R-group).
●● Proline Ramachandran Plot
Answer: Proline adopts fewer angles. Amino group is incorporated into a
ring.
●● α-helices
Answer: Ala is common, Gly & Pro are not very common. Side-chain
interactions every 3 or 4 residues. Turns once every 3.6 residues.
Distance between backbones is 5.4Å.
●● Helix Dipole
Answer: Formed from added dipole moments of all hydrogen bonds in
an α-helix. N-terminus is δ+ and C-terminus is δ-.
●● ß-sheet
Answer: Either parallel or anti-parallel. Often twisted to increase
strength.
●● Anti-parallel ß-sheet
Answer: Alternating sheet directions (C & N-termini don't line-up). Has
straight H-bonds.
QUESTIONS AND CORRECT ANSWERS
●● FMOC Chemical Synthesis
Answer: Used in synthesis of a growing amino acid chain to a
polystyrene bead. FMOC is used as a protecting group on the N-
terminus.
●● Salting Out (Purification)
Answer: Changes soluble protein to solid precipitate. Protein precipitates
when the charges on the protein match the charges in the solution.
●● Size-Exclusion Chromatography
Answer: Separates sample based on size with smaller molecules eluting
later.
●● Ion-Exchange Chromatography
Answer: Separates sample based on charge. CM attracts +, DEAE
attracts -. May have repulsion effect on like charges. Salt or acid used to
remove stuck proteins.
●● Hydrophobic/Reverse Phase Chromatography
,Answer: Beads are coated with a carbon chain. Hydrophobic proteins
stick better. Elute with non-H-bonding solvent (acetonitrile).
●● Affinity Chromatography
Answer: Attach a ligand that binds a protein to a bead. Elute with harsh
chemicals or similar ligand.
●● SDS-PAGE
Answer: Uses SDS. Gel is made from cross-linked polyacrylamide.
Separates based off of mass with smaller molecules moving faster.
Visualized with Coomassie blue.
●● SDS
Answer: Sodium dodecyl sulfate. Unfolds proteins and gives them
uniform negative charge.
●● Isoelectric Focusing
Answer: Variation of gel electrophoresis where protein charge matters.
Involves electrodes and pH gradient. Protein stops at their pI when
neutral.
●● FDNB (1-fluoro-2,3-dinitrobenzene)
,Answer: FDNB reacts with the N-terminus of the protein to produce a
2,4-dinitrophenol derivative that labels the first residue. Can repeat
hydrolysis to determine sequential amino acids.
●● DTT (dithiothreitol)
Answer: Reduces disulfide bonds.
●● Iodoacetate
Answer: Adds carboxymethyl group on free -SH groups. Blocks
disulfide bonding.
●● Homologs
Answer: Shares 25% identity with another gene
●● Orthologs
Answer: Similar genes in different organisms
●● Paralogs
Answer: Similar "paired" genes in the same organism
●● Ramachandran Plot
Answer: Shows favorable phi-psi angle combinations. 3 main "wells"
for α-helices, ß-sheets, and left-handed α-helices.
, ●● Glycine Ramachandran Plot
Answer: Glycine can adopt more angles. (H's for R-group).
●● Proline Ramachandran Plot
Answer: Proline adopts fewer angles. Amino group is incorporated into a
ring.
●● α-helices
Answer: Ala is common, Gly & Pro are not very common. Side-chain
interactions every 3 or 4 residues. Turns once every 3.6 residues.
Distance between backbones is 5.4Å.
●● Helix Dipole
Answer: Formed from added dipole moments of all hydrogen bonds in
an α-helix. N-terminus is δ+ and C-terminus is δ-.
●● ß-sheet
Answer: Either parallel or anti-parallel. Often twisted to increase
strength.
●● Anti-parallel ß-sheet
Answer: Alternating sheet directions (C & N-termini don't line-up). Has
straight H-bonds.
