Affinity Chromatography - Department of Molecular and Cellular ...

Sample: Pooled and frozen human plasma from 5 donors. 50 ml of thawed plasma filtered
(0.45 µM). Plasma and binding buffer mixed in ratio 2:1
Binding buffer: 0.1 M Tris-HCl, 0.01 M citric acid, 0.225 M NaCl, pH 7.4
Wash buffer: 0.1 M Tris-HCl, 0.01 M citric acid, 0.330 M NaCl, pH 7.4
Elution buffer: 0.1 M Tris-HCl, 0.01 M citric acid, 2.0 M NaCl, pH 7.4
Column: Heparin Sepharose 6 Fast Flow packed in HR 5/5 column
Chromatographic
procedure:
5 ml binding buffer, 45 ml sample, 40 ml binding buffer,
15 ml wash buffer, 9 ml elution buffer
2
A 280 nm
1 2 3 4 5 6 7 8
1
0
Wash Elution
0 50 100 150 200 250
B
C
Time (min)
Isoelectric focusing-PAGE analysis of the peaks B
and C from the affinity chromatography.
Lanes 1 and 4. Peak C
Lanes 2 and 7. IEF calibration kit
Lanes 3 and 6. Antithrombin-III from
Sigma (A7388)
Lanes 5 and 8. Peak B
The results show that pure antithrombin-III is
present in the two peaks. NOR-Partigen-
Antithrombin-III test of peaks B and C shows a
more active form of antithrombin-III concentrated
in peak C.
Fig. 38. Purification of antithrombin-III from human plasma on Heparin Sepharose 6 Fast Flow. Peak B elutes with
wash buffer. Peak C elutes with elution buffer and includes a more active form of antithrombin-III.
Performing a separation
As for DNA binding proteins, see page 62.
Since the heparin acts as an affinity ligand for coagulation factors, it may be advisable to
include a minimum concentration of 0.15 M NaCl in the binding buffer.
If an increasing salt gradient gives unsatisfactory results, use heparin (1–5 mg/ml) as a
competing agent in the elution buffer.
Biotin and biotinylated substances
HiTrap Streptavidin HP, Streptavidin Sepharose High Performance
Biotin and biotinylated substances bind to streptavidin (a molecule isolated from Streptomyces
avidinii) in a very strong interaction that requires denaturing conditions for elution.
By coupling streptavidin to Sepharose a highly specific affinity medium is created and,
using biotinylated antibodies, the strong interaction can be utilized for the purification of
antigens. The biotinylated antibody-antigen complexes bind tightly to Streptavidin
Sepharose and the antigen can then be eluted separately using milder elution conditions,
leaving behind the biotinylated antibody. An alternative to labelling the antibody with
biotin is to use 2-iminobiotin that binds to streptavidin above pH 9.5 and can be eluted at
pH 4 (see Figure 39).
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