Mirco Lab Midterm: Lecture 5 Flashcards
What are the principles of chromatography
- Chromatography is a method for separation that
describes separation of solutes by different partitioning
between two phases - One of the phases is immobile (stationary): as used in
molecular biology, in most cases it is solid - The second phase is mobile (flowing) phase: in
molecular biology in most cases it is liquid - The chromatography process results in different
mobility of solutes (down a column or a layer of solid
stationary phase) in the presence of flowing phase
What is Partitioning coefficient - α?
It is defined as the fraction of the solute that is adsorbed on the
solid phase
- Mathematically, the partitioning coefficient α = [adsorbed
solute]/[total (adsorbed + desorbed) solute] - One can determine α of a solute by measuring its rate of
movement relative to the liquid flow (Rf
): Rf = 1-α and assuming
that the solute equilibrates rapidly between the stationary and
mobile phases
Example: solute with a partitioning coefficient 0.9 moves at
10% of the speed of the liquid flow or (1-α), because at each
instance 90% of it is adsorbed to the stationary phase and only
10% is free in solution
Types of chromatography used in protein
separation
Column chromatography (regular – low pressure,
HPLC)/batch adsorption
- Ion exchange
- Inorganic adsorbents (bentonite, hydroxyapatite,
titanum oxide) - Hydrophobic chromatography
- Gel filtration – size exclusion chromatography
- Affinity chromatography (immobilized metal
affinity, ligands, antibodies, dye-ligands
What are the stationary phase materials?
Cellulose
- Agarose
- Synthetic polymers
- Inorganic materials (HAP, glass)
- Magnetic beads
- Plastic surfaces, membranes, array surfaces
- Micro-beads, macro-beads, nanoparticles
Describe the protein binding to the stationary phase
(adsorbent, matrix)
There is no simple dissociation equilibrium
constant describing protein binding to the matrix.
In most cases however, one can use a single
“average” or “apparent” dissociation constant
- Kd
= m*p/q where m: concentration of free (nonoccupied by protein) binding sites on the matrix;
p: concentration of free protein in solution at
equilibrium with the matrix; q: concentration of
matrix-bound protein
How does the chromatography work?
- For α = 0 there is no adsorption
- For α = 1 all protein is adsorbed
- For 0<α<1 protein adsorbs partially and moves
down the column, emerging when 1/1-α
column volumes of liquid phase passed
through the column - Practically α must be ≥0.8 for an useful
adsorption
What is affinity chromatography?
Separates proteins based on reversible interaction between the protein
and specific ligand coupled to a chromatography matrix
- Highly selective – the purification can be in the order of several thousandfold in a single step, with high capacity and recovery of active protein
- The only chromatography that enables purification of proteins on the
basis of their biological function or individual structure. - Used to separate active proteins from their denatured or functionally
different forms - Pure proteins can be isolated from crude samples even if present at very
low concentration - Can be single protein specific (purification of recombinant fusion
proteins) or specific for a class of proteins i.e. nucleic acids binding
proteins
What is affinity chromatography matrix
- Ligand must be attached covalently to the
matrix - Ligand must be attached in a way that does
not prevent protein binding (spacer arm
keeping the ligand away from the matrix to
prevent steric interference) - Minimal non specific interactions
Immobilization (coupling) of the ligands to
the solid support (matrix)
Covalent chemical bonding between particular functional
groups of the ligand (primary amines, sulfhydryls, carboxylic
acids, aldehydes) and reactive groups of the matrix
- To facilitate the binding coupling might be done by orienting
of the ligands on the surface of the matrix so that proteins
can easily recognize them - Covalent coupling must be stable (no leakage of ligand
during the binding, washes, elution), prevent non-specific
binding, prevent modification of the ligand that will lower its
binding potential, prevent over cross-linking of the resin
(matrix)
Application of the chromatography theory to
affinity chromatography
- The dissociation equilibrium constant of protein
binding to the affinity adsorbent should be
similar to the dissociation equilibrium constant of
interaction between the protein and free ligand - The binding capacity of a typical affinity matrix
(1-5 mg/ml of protein) translates to mt
value of
0.01-0.05mM - Typical concentration of protein to be purified is
≤ 0.01mM
Non-specific interactions contribute to the
free energy of binding
- Ligands attached to matrixes via hydrophobic “spacer
arms”, but not via hydrophylic “spacer arms” - Non-specific, hydrophobic interactions with the spacer
arms contribute to the total free energy of protein binding
to the matrix - These interactions provide additional (to the specific
binding) free energy of binding, that stabilizes (drives)
protein-ligand binding to the matrix. Binding decreases
either in the absence of specific interactions (i.e. when
there is excess of free ligand) or without non-specific
interactions (i.e. hydrophylic spacer arms)
Why is affinity chromatography is a powerful
method for protein purification
Frequently can purify proteins from complex mixes
more than 1000 X with high recovery in one step
- Protein binding and washes are done in mild
conditions - Proteins are eluted by: 1. Competition with free
ligand (α drops to zero so the protein dissociates
from the ligand); 2. For strong interactions harsher
elution with high salt or change of pH are used (i.e.
antibody/antigen immuno-purification)
What are the practical steps in affinity chromatography?
- Selection of chromatography media: preactivated, ready for covalent coupling of a
ligand or media with coupled ligand - Selection of format: pre-packed columns, batch
mode - Purification method: optimal flow rate (must be
optimized to achieve efficient binding and to
maximize the recovery during protein elution) - Equilibration of the media with binding buffer
- Sample application and wash: sample volume does
not affect the separation since AC is a binding
technique; for weak affinity or slow equilibration a
low flow rate might be required - Elution: change ionic strength of the elution buffer,
change the pH (usually low or high extremes: could
inactivate the eluted protein); competitive elution:
the elution agent competes either for binding to
target protein or to the ligand
What is batch adsorption?
Very easy, convenient, does not require
columns
- However it has an essential requirement: α of the
protein to the ligand coupled to the matrix has to be
very close to 1 (meaning very strong interaction) while
all other proteins must have very low α - The reason is that the concentration of free protein is
the same throughout the volume of sample + matrix,
so the total amount of not adsorbed protein will be
substantial if α is not very close to 1
What is GST fusion proteins affinity purification?
Affinity resin of glutathione immobilized to
crosslinked agarose (sepharose), magnetic
beads
- Binding in mild conditions in the presence of
non-ionic detergents - Elution with excess of reduced glutathione
- Removal of the GST tag by enzymatic cleavage
with thrombin, Factor Xa
