BCH4024 EXAM 1 QUESTIONS
WITH CORRECT DETAILED
ANSWERS
Size/shape method of seperation - Answer-size-exclusion chromatography
Charge (pI) method of seperation - Answer-Ion exchange chromatography
Binding properties method of seperation - Answer-Affinity chromatography
Basic protein purification steps - Answer-1) Lyse cell to release soluble proteins (crude
extract)
-Sonicator: sound waves
-French press: pressure
2) Centrifuge to separate.
-Insoluble materials form pellet
-Soluble proteins can be poured off and analyzed (soluble lysate)
3) Continue to separate.. (possibly w ammonium sulfate precipitation)
Ammonium sulfate precipitation procedure - Answer-1) precipitate and remove
undesired proteins
Add low [salt] --> centrifuge to pellet precipitate --> discard pellet/precipitate
2) Precipitate and isolate desired protein
Increase [salt] --> centrifuge to pellet precipitate --> discard supernatant
Low [Salt] - Answer-Strong attractive force between proteins
Optimal [Salt] - Answer-Salt ions "shield" charges on proteins so weak attractive force
High [Salt] - Answer-Ions compete for water
Column Chromatography - Answer-Protein solution is poured over the stationary phase
followed by a buffered solution being pumped through
Ion-Exchange Chromatography - Answer-Separation based on charge
Cation exchanger (-resin) binds cation
Anion exchanger (+resin) binds anions
Protein w smaller charges elute first
, Factors Effecting Ion Exchange Chromatography - Answer--Net charge on molecule
-Solution pH
-Ionic strength
size exclusion chromatography - Answer-Small protein can diffuse in and out of the
pores of the resin increasing distance traveled. Large proteins cannot penetrate pores
as easily
Affinity Chromatography - Answer-Proteins bind to resin via specific interactions with
ligands covalently bound to the resin. Unbound proteins travel through 1st
Gel electrophoresis - Answer-analytical technique, measures migration in an electric
field. How fast protein moves depends on:
electrophoretic mobility
velocity
electrical potential
net charge
frictional coefficient
Electrophoresis using SDS - Answer-denatures and coats the protein with SDS to give
the protein a highly negative charge. 1 SDS for every 2 AA's. Separation depends on
mass
Isoelectric Focusing - Answer-Used to determine the pI by monitoring a proteins
movement across a pH gradient. When pI=pH, net charge is 0 and protein will stop
Two-dimensional Electrophoresis - Answer-combination of isoelectric focusing and
SDS-PAGE
Myoglobin and Hemoglobin - Answer-oxygen-binding heme proteins
Myoglobin - Answer-intracellular storage in the muscles
Hemoglobin - Answer-used for transport with red blood cells from lungs to tissues
Why is O2 transport necessary? - Answer-Oxygen is poorly soluble in aqueous solution
and diffusion through tissue is effective for only a few mm's
Structure of Myoglobin - Answer-1 polypeptide chain w 153 amino acids residues and 1
heme and its Fe2+ group
78% residues found in 1 of 8 right-handed a-helices
Hydrophobic pocket protects heme group
Properties of Myoglobin - Answer--Binds reversibly to oxygen so it can be released if
needed
WITH CORRECT DETAILED
ANSWERS
Size/shape method of seperation - Answer-size-exclusion chromatography
Charge (pI) method of seperation - Answer-Ion exchange chromatography
Binding properties method of seperation - Answer-Affinity chromatography
Basic protein purification steps - Answer-1) Lyse cell to release soluble proteins (crude
extract)
-Sonicator: sound waves
-French press: pressure
2) Centrifuge to separate.
-Insoluble materials form pellet
-Soluble proteins can be poured off and analyzed (soluble lysate)
3) Continue to separate.. (possibly w ammonium sulfate precipitation)
Ammonium sulfate precipitation procedure - Answer-1) precipitate and remove
undesired proteins
Add low [salt] --> centrifuge to pellet precipitate --> discard pellet/precipitate
2) Precipitate and isolate desired protein
Increase [salt] --> centrifuge to pellet precipitate --> discard supernatant
Low [Salt] - Answer-Strong attractive force between proteins
Optimal [Salt] - Answer-Salt ions "shield" charges on proteins so weak attractive force
High [Salt] - Answer-Ions compete for water
Column Chromatography - Answer-Protein solution is poured over the stationary phase
followed by a buffered solution being pumped through
Ion-Exchange Chromatography - Answer-Separation based on charge
Cation exchanger (-resin) binds cation
Anion exchanger (+resin) binds anions
Protein w smaller charges elute first
, Factors Effecting Ion Exchange Chromatography - Answer--Net charge on molecule
-Solution pH
-Ionic strength
size exclusion chromatography - Answer-Small protein can diffuse in and out of the
pores of the resin increasing distance traveled. Large proteins cannot penetrate pores
as easily
Affinity Chromatography - Answer-Proteins bind to resin via specific interactions with
ligands covalently bound to the resin. Unbound proteins travel through 1st
Gel electrophoresis - Answer-analytical technique, measures migration in an electric
field. How fast protein moves depends on:
electrophoretic mobility
velocity
electrical potential
net charge
frictional coefficient
Electrophoresis using SDS - Answer-denatures and coats the protein with SDS to give
the protein a highly negative charge. 1 SDS for every 2 AA's. Separation depends on
mass
Isoelectric Focusing - Answer-Used to determine the pI by monitoring a proteins
movement across a pH gradient. When pI=pH, net charge is 0 and protein will stop
Two-dimensional Electrophoresis - Answer-combination of isoelectric focusing and
SDS-PAGE
Myoglobin and Hemoglobin - Answer-oxygen-binding heme proteins
Myoglobin - Answer-intracellular storage in the muscles
Hemoglobin - Answer-used for transport with red blood cells from lungs to tissues
Why is O2 transport necessary? - Answer-Oxygen is poorly soluble in aqueous solution
and diffusion through tissue is effective for only a few mm's
Structure of Myoglobin - Answer-1 polypeptide chain w 153 amino acids residues and 1
heme and its Fe2+ group
78% residues found in 1 of 8 right-handed a-helices
Hydrophobic pocket protects heme group
Properties of Myoglobin - Answer--Binds reversibly to oxygen so it can be released if
needed
