Affinity Chromatography - Department of Molecular and Cellular ...

Sample: 2000 ml partially purified sample from
DEAE Sepharose CL-4B flow-through,
pH 7.0
Column: HiPrep 16/10 Heparin FF
Binding buffer: 50 mM sodium phosphate, pH 7.5
Elution buffer: 50 mM sodium phosphate,
1 M sodium chloride, pH 7.5
Flow:
1.5 ml/min (45 cm/h)
Chromatographic
procedure:
A ( )
Equilibration binding buffer: 80 ml
Sample application: 2000 ml
Wash with binding buffer: 100 ml
Elution: 300 ml elution buffer as
linear gradient 0–100%
1 1
fr. 13–14
280
fr. 23–24
scCro8, fr. 54–55
fr. 69
( ) [NaCl] (M)
M r
97 000
66 000
45 000
30 000
20 100
14 400
1 2 3 4 5 6 7 8 9 10
Electrophoresis: SDS-PAGE, 12% gel, Coomassie Blue staining
Lane 1. Pool from HiPrep 26/10 Desalting
Lane 2. Flow-through pool from DEAE Sepharose CL-4B
Lane 3. Low Molecular Weight Calibration Kit (LMW)
Lane 4-10. Eluted fractions from HiPrep 16/10 Heparin FF
Lane 4. Fraction 13
Lane 5. Fraction 14
Lane 6. Fraction 23
Lane 7. Fraction 24
Lane 8. Fraction 54
Lane 9. Fraction 55
Lane 10. Fraction 69
0 0
Fig. 36. scCro8 purification on HiPrep 16/10 Heparin FF.
Performing a separation
Binding buffers: 20 mM Tris-HCl, pH 8.0 or 10 mM sodium phosphate, pH 7.0
Elution buffer: 20 mM Tris-HCl, 1–2 M NaCl, pH 8.0 or 10 mM sodium phosphate, 1–2 M NaCl, pH 7.0
1. Equilibrate the column with 10 column volumes of binding buffer.
2. Apply the sample.
3. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by
UV absorption at A 280 nm ).
4. Elute with 5–10 column volumes of elution buffer using a continuous or step gradient from 0–100% elution buffer.
Modify the selectivity of heparin by altering pH or ionic strength of the buffers. Elute using
a continuous or step gradient with NaCl, KCl or (NH 4 ) 2 SO 4 up to 1.5–2 M.
Cleaning
Remove ionically bound proteins by washing with 0.5 column volume 2 M NaCl for
10–15 minutes.
Remove precipitated or denatured proteins by washing with 4 column volumes 0.1 M NaOH
for 1–2 hours or 2 column volumes 6 M guanidine hydrochloride for 30–60 minutes or
2 column volumes 8 M urea for 30–60 minutes.
Remove hydrophobically bound proteins by washing with 4 column volumes 0.1% – 0.5%
Triton X-100 for 1–2 hours.
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