Affinity Chromatography - Department of Molecular and Cellular ...

A 280 nm
1 2 3 4 5
Purification options
Binding capacity Maximum Comments
operating flow
HiTrap Bovine antithrombin III, 3 mg/column 4 ml/min (1 ml column) Prepacked columns.
Heparin HP Bovine antithrombin III, 15 mg/column 20 ml/min (5 ml column)
HiPrep 16/10 Bovine antithrombin III, 40 mg/column 10 ml/min Prepacked 20 ml column.
Heparin FF
Heparin Bovine antithrombin III, 2 mg/ml medium 400 cm/h* Supplied as a suspension
Sepharose 6
ready for column packing.
Fast Flow
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from
measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Purification examples
Figures 34, 35 and 36 show examples of conditions used for the purification of different
DNA binding proteins.
Sample:
49 ml E. coli lysate (= 1 g cells) after passage through a
5 ml DEAE Sepharose Fast Flow column
Column:
HiTrap Heparin HP, 1 ml
Flow:
1.0 ml/min
Binding buffer: 20 mM Tris-HCl, 1 mM EDTA, 1 mM 2-mercaptoethanol,
2% glycerol, pH 8.0
Elution buffer: Binding buffer + 1.0 M NaCl
Elution conditions: 25 ml elution buffer, linear gradient 0–100%
M r
97 000
66 000
45 000
30 000
20 100
14 400
0.4
0.3
0.2
0.1
% Elution buffer
100
50
SDS-PAGE, PhastSystem, PhastGel Gradient
8–25, 1 ml sample, silver stained.
Lane 1. Weight (LMW) calibration kit,
reduced
Lane 2. Reverse transciptase, reduced
Lane 3. Pool I from HiTrap Heparin HP,
1 ml reduced
Lane 4. Unbound material from
DEAE Sepharose FF, reduced
Lane 5. Cell lysate, reduced
pool I
0
6
50
60
70
80 ml
Fig. 34. Partial purification of recombinant HIV-reverse transcriptase on HiTrap Heparin HP.
60