Biochem 301 Exam 3 with
100% correct answers
(graded A+)
Bulk techniques for protein separation - answer Charge, solubility,
thermal stability
Chromatography techniques for protein separation - answer Charge,
size, affinity for a ligand, hydrophobicity
Classical biochemistry - answer Pull one enzyme out a mixture
Modern day biochemistry - answer Look at everything at once:
genes - nucleic acids - all proteins at once
Salt precipitation - answer At high salt concentrations, the solubility
of proteins tends to decrease as water molecules become bound to
salt ions (high dielectric constant) reducing protein solubilization.
Column chromatography - answer Chromatography in which the
substances to be separated are introduced onto the top of a column
packed with an adsorbent (as silica gel or alumina), pass through
the column at different rates that depend on the affinity of each
substance for the adsorbent and for the solvent or solvent mixture,
and are usually collected in solution as they pass from the column
at different times
ion exchange chromatography - answer Depending on resin,
different attractions occur.
Stationary phase is made of charged beads which attract
compounds of opposite charge.
,Depends on net charge of protein (based on individual aa charges)
Start at low salt (everything attracted) increase salt concentration
to loosen attraction, bump things out that are more weakly bound.
size exclusion chromatography (gel filtration) - answer -beads
contain tiny pores
-small molecules enter the pores & get stuck so they elude later
-large molecules don't fit in the pores so they move around taking a
shorter path & elude faster in void volume
affinity chromatography - answer beads are coated w/ nickel that
binds the protein of interest (due to addition of multiple his tags,
don't naturally occur)
can be eluted by washing the column w/ imidazole which competes
for nickel binding
Electrophoresis - answer Method of separating proteins by size,
most commonly SDS PAGE via electric field (all negatively charged
because coated with SDS)
Small proteins move quickly, large move slowly
Good for crude estimate of size
isoelectric focusing - answer A specialized method of separating
proteins by their isoelectric point using electrophoresis; the gel is
modified to possess a pH gradient.
, 2D electrophoresis - answer a type of electrophoresis that is the
combination of SDS-PAGE and isoelectric focusing; starts with PI
(horses lining up to race) then uses SDS PAGE
specific activity (enzyme) - answer The activity of an enzyme per
milligram of total protein (expressed in ìmol/time/mg of all protein).
Gives a measurement of enzyme purity in the mixture.
It is the micromoles of product formed by an enzyme in a given
amount of time (milliseconds, seconds, minutes) under given
conditions per milligram of total protein.
Spectroscopic Detection of Aromatic Amino Acids - answer Trp and
Tyr are strong chromophores, if protein contains these you can use
this method.
ELISA - answer Enzyme-linked immunosorbent assay
How much of a particular protein exists in a mixture
Uses enzyme conjugated to an antibody to catalyze a colorimetric or
fluorescent reaction
Direct ELISA - answer detecting protein directly by using a primary
antibody that is enzyme-linked
Pros: quick, one antibody; no cross reactivity because only one ab
Cons: Expensive since specific linked ab necessary for each ELISA
100% correct answers
(graded A+)
Bulk techniques for protein separation - answer Charge, solubility,
thermal stability
Chromatography techniques for protein separation - answer Charge,
size, affinity for a ligand, hydrophobicity
Classical biochemistry - answer Pull one enzyme out a mixture
Modern day biochemistry - answer Look at everything at once:
genes - nucleic acids - all proteins at once
Salt precipitation - answer At high salt concentrations, the solubility
of proteins tends to decrease as water molecules become bound to
salt ions (high dielectric constant) reducing protein solubilization.
Column chromatography - answer Chromatography in which the
substances to be separated are introduced onto the top of a column
packed with an adsorbent (as silica gel or alumina), pass through
the column at different rates that depend on the affinity of each
substance for the adsorbent and for the solvent or solvent mixture,
and are usually collected in solution as they pass from the column
at different times
ion exchange chromatography - answer Depending on resin,
different attractions occur.
Stationary phase is made of charged beads which attract
compounds of opposite charge.
,Depends on net charge of protein (based on individual aa charges)
Start at low salt (everything attracted) increase salt concentration
to loosen attraction, bump things out that are more weakly bound.
size exclusion chromatography (gel filtration) - answer -beads
contain tiny pores
-small molecules enter the pores & get stuck so they elude later
-large molecules don't fit in the pores so they move around taking a
shorter path & elude faster in void volume
affinity chromatography - answer beads are coated w/ nickel that
binds the protein of interest (due to addition of multiple his tags,
don't naturally occur)
can be eluted by washing the column w/ imidazole which competes
for nickel binding
Electrophoresis - answer Method of separating proteins by size,
most commonly SDS PAGE via electric field (all negatively charged
because coated with SDS)
Small proteins move quickly, large move slowly
Good for crude estimate of size
isoelectric focusing - answer A specialized method of separating
proteins by their isoelectric point using electrophoresis; the gel is
modified to possess a pH gradient.
, 2D electrophoresis - answer a type of electrophoresis that is the
combination of SDS-PAGE and isoelectric focusing; starts with PI
(horses lining up to race) then uses SDS PAGE
specific activity (enzyme) - answer The activity of an enzyme per
milligram of total protein (expressed in ìmol/time/mg of all protein).
Gives a measurement of enzyme purity in the mixture.
It is the micromoles of product formed by an enzyme in a given
amount of time (milliseconds, seconds, minutes) under given
conditions per milligram of total protein.
Spectroscopic Detection of Aromatic Amino Acids - answer Trp and
Tyr are strong chromophores, if protein contains these you can use
this method.
ELISA - answer Enzyme-linked immunosorbent assay
How much of a particular protein exists in a mixture
Uses enzyme conjugated to an antibody to catalyze a colorimetric or
fluorescent reaction
Direct ELISA - answer detecting protein directly by using a primary
antibody that is enzyme-linked
Pros: quick, one antibody; no cross reactivity because only one ab
Cons: Expensive since specific linked ab necessary for each ELISA
