Download Affinity Chromatography

Separation techniques
?
Molecules can be separated:
Chemically: by charge, by action with
specific reagents
Physically: by solubility, by molecular
weight, by density, structure (e.g.
shape)
Separation
1. By physical properties
- Centrifugation (centrifugal force)
- SDS-polyacrylamid-gellectrophoresis (charge)
- Agarose-gellectrophoresis (charge)
- Gelfiltration (molecular mass)
2. By chemical properties
- Ion exchange chromatography
- Affinity chromatography
- Solubility/desalting
- DNA/protein chips or microarrays
3. By physical-chemical properties
Native polyacrylamid-gelelectrophoresis
Paper-electrophoresis
2D-gelelectrophoresis
Centrifugation
1. Differential centrifugation
- Pellet/supernatant
2. Density gradient centrifugation
- Continuous gradient
- Step gradient
Gel filtration or
size exclusion chromatography
Approximately half of the volume of GFC columns is occupied by the resin. Small
molecules, which enter all the pores of the resin elute after one column volume.
For large molecules, which can not enter any of the pores, at least half of the
column volume is necessary. These molecules leave the column after half
column volume.
Intermediate sized molecules can enter some of the pores, but not all – they elute
between the two other fractions. Ball shaped (isometric) molecules are well
separated, elongated ones are difficult to separate. Why?
Gel filtration does not show
good separation, but can
be used for desalting or for
the separation of molecules
of very different size.
Agarose gel electrophoresis
agarose
Agarose is a polysacharid extracted and
purified from sea moss. For separation of large
molecules, such as proteins or DNA.
DNA stain
Agarose gel electrophoresis
Pulse field gel electrophoresis
Continuously changing the direction
of the electric field. The large
charged particles will not be
trapped in the network of
polyacrylamide. Very large
molecules and particles, even
chromosomes can be separated
by this technique.
The picture shows the separation of
chromosomes from different
strains of Plasmodium, the
parasite causing disease malaria.
Polyacrylamide electrophoresis (PAGE)
For separaton of small molecules, such as proteins or small DNA fragments.
Native-PAGE
SDS-PAGE
- Molecule mass
- Charge
- Molecule mass
+ SDS (sodium dodecyl sulphate)
+ reducing agent (e.g. mercaptoethanol)
- No charge difference between molecules
- No secondary structure (with reducing agent)
Paper electrophoresis
Electrophoresis of proteins
can be carried out on wet
paper. Though paper
electrophoresis does not
have a good resolution.
However, it is a valuable
tool, e.g. it can be used for
the separation of native
proteins. (in SDS gel
electrophoresis the proteins
are in denatured state).
On this figure we see
electrophoretograms of
healthy and sick people.
Isoelectric focusing
separation on pH-gradient by the charge
A
B
P
h
g
r
a
d
i
e
n
t
-
9
Forming a pH gradient:
e.g.A With free amfolins or
immobilens (weak acidic or
basic molecules)
8
7
6
Amfolins - forms a pH
e.g.B gradient in electronic field
5
4
+
Immobilens -covalently
bound to the polyacrylamide
matrix
2D-electrophoresis
First dimension:
Isoelectric focusing
Proteins in a breast cancer
Second dimension:
Separation by molecule mass
Proteins in a healthy breast
Ion exchange chromatography of proteins
At slightly basic pH (8-9) most proteins are negatively charged and bind
to anionic exchangers. When the ion
concentration is raised, first those
molecules are eluted which have
the lowest number of negative side
chains. Highly negatively charged
proteins can be eluted only with
high ionic strength solutes.
The figure shows that fractions eluting
at different salt concentrations
contain very different proteins.
Ion exchange chromatography
ANIONS with exchangeable counterions
starting
adsorbtion
buffer counterions
CATIONS with exchangeable counterions
start desorbtion end desorbtion
separable materials
regeneration
gradient ions
Affinity chromatography
Affinity chromatography is a powerful
technique to purify one component
out of highly complex mixtures. It is
based on the interaction of two
molecules (biotin-avidin). One is fixed
on the column, while the other is
selected out of the mixture.
A special form of affinity chromatography
is metal chelate chromatography,
where complexes of heavy metal ions
are fixed on the column. These
columns have high affinity to proteins
or peptides with numerous histidine
residues. Elution is carried out by
imidazol compounds (His is imidazolalanine).
Affinity Chromatography
immobilization
Sample (S) adsorption
impurities
Desorption of the sample
Affinity separations
Affinity chromatography frequently uses antibodies. Another separation
technique, which is based on the use of antibodies is magnetic
separation. Proteins, organelles, even intact living cells can be
separated with colloidal, plastic-coated magnetic beads, carrying
antibodies on their surface. The figure illustrates the principle of the
separation:
mRNA can also separated with Dynabeads coated with oligo(dT) primers. Why?
DNA/Protein chips or microarrays
For genome wide analysis.
DNA chips contains single stranded DNA spots on
a glass surface, capable for hybridization with the
complementary strand of a cDNA clone reverse
transcribed from a single mRNA. One spot
represents one gene. There can be 50 000 spots
or more on a glass surface. The whole genome
can be screened => Genomics
In protein chips
there are antibodies
bound to the glass
surface.
Each spot
recognizes different
protein molecules. The
whole
Proteome can be
screened.
=>Proteomics