Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Affinity Chromatography
separates proteins on the basis of the ability of certain proteins to bind
certain other molecules (ligands)
Size Exclusion Chromatography
separates proteins on the basis of their size.
sophisticated instrumentation
and columns
mainly
soluble proteins
insoluble cell
components
Affinity chromatography
monitor successive
purification at
each step using gel
electrophoresis and
protein staining
10.79
8.95
pKacarboxyl = 2.18
pKaamino = 8.95
pKasidechain = 10.79
10.79
8.95
Nelson p85
(pK2 + pK3)
pI =
(10.79 + 8.95)
=
= 9.87
**of course a minority of proteins will have an pI = 7 and they will be neutral
at pH 7.
Protein 2
17
Lysine
Aspartate
Glutamate
Net charge
+15
-5
Arginine
These net charges can help purify a target protein away from other cellular proteins
using ion-exchange chromatography.
A chromatography column
Positively
charged
medium
flow of buffer
Proteins bound to beads can be released by adding other negative charged ions
that will compete with protein for binding the beads
NaCl is used because the negatively charged Chloride ions will act as competitor
Cl
Na+
Cl
Na+
Na+
Cl
Cl
Na+
Cl
Na+
Cl
This is why
DEAE is called
an ANION
exchange medium
Affinity Chromatography
affinity chromatography relies on fact that proteins, as part of their function, bind
other molecules called Ligands.
For example, some proteins that help regulate gene expression bind double-stranded
DNA at a specific sequence of nucleotides.
If you are trying to purify such a protein, it would be easy to make your own
affinity chromatography medium.
Bead
Ligand
DNA
small
protein
medium
protein
large
protein
flow of buffer
amount of protein
20 kDa
100 kDa
40 kDa
time
mainly
soluble proteins
insoluble cell
components
Affinity chromatography
monitor successive
purification at
each step using gel
electrophoresis and
protein staining
+
stain the gel to
visualize proteins
Proteins are treated with detergent SDS (sodium dodecyl sulfate) and a reducing agent
before running on gel
structure of SDS
Proteins are treated with detergent SDS (sodium dodecyl sulfate) and a reducing agent
before running on gel
SDS
Beta-mercaptoethanol
(reducing agent)
S S
Reminder
Midterm 1 Wed Oct 14