Affinity Chromatography - Department of Molecular and Cellular ...

Performing a separation
Binding buffer: 20 mM Tris-HCl, 0.1–0.5 M NaCl, pH 7.0.
If required, include 8 M urea or 6 M guanidine HCl to ensure that the protein is denatured and all thiol groups
are accessible for the reaction.
1 mM EDTA can be added to remove trace amounts of catalytic heavy metals.
Elution buffer alternatives:
For covalently bound proteins: 0.025 M cysteine, 50 mM Tris-HCl, pH 7–8.
To minimize reduction of intramolecular disulphide bridges: 5–20 mM L-cysteine, 50 mM Tris-HCl, 1 mM EDTA,
pH 8.0 or 20–50 mM 2-mercaptoethanol, 50 mM Tris-HCl, 1 mM EDTA, pH 8.0.
Note: When using Thiopropyl Sepharose, 2-thiopyridyl groups must be removed after the protein has bound.
Wash the column with sodium acetate 0.1 M, 2-mercaptoethanol 5 mM, pH 4.0 before beginning elution.
N.B. Degas all buffers to avoid oxidation of free thiol groups.
If the proteins to be purified contain disulphide bonds, the disulphide bridges must be
reduced, for example with 2-mercaptoethanol, (5 mM).
Analyse the thiol content of the sample by thiol titration to ensure that the capacity of the
medium will not be exceeded.
Use preliminary titration studies with 2,2'-dipyridyl disulphide to provide a guide to
optimal coupling conditions. A spectrophotometer can be used to determine the release of
2-thiopyridone (absorbance coefficient = 8.08 x 10 3 M -1 cm -1 at 343 nm) when the sample
(1–5 mg in 1–3 ml binding buffer) reacts with 2, 2'-dipyridyl disulphide. Choose the
conditions to suit the specific sample. Under standard conditions at pH 7.5, a few minutes
is usually enough for a complete reaction.
1. Use a desalting column to transfer pre-dissolved sample into the binding buffer (see page 134) and to remove
any low molecular weight thiol compounds and reducing agents that might interfere with the coupling reaction.
2. Weigh out the required amount of powder (1 g gives about 3 ml for Activated Thiol Sepharose 4B and 4 ml
for Thiopropyl Sepharose 6B).
3. Wash and re-swell on a sintered glass filter (porosity G3), using degassed, distilled water or binding buffer
(200 ml/g, 15 min at room temperature) to remove additives.
4. Prepare the slurry with binding buffer in a ratio of 75% settled medium to 25% buffer.
5. Pack the column (see Appendix 3) and equilibrate with binding buffer.
6. Load the sample at a low flow (5–10 cm/h) and leave in contact with the medium for at least one hour to
ensure maximum binding.
7. Wash the column with binding buffer until no material appears in the eluent (monitored by UV absorption
at A 280 nm ).
8. Elute the target molecules with elution buffer using a low flow (5–10 cm/h).
The coupling reaction can be monitored and, in some cases, quantified by following the
appearance of 2-thiopyridone in the eluent at 343 nm during the purification.
Sodium phosphate or ammonium acetate can be used as an alternative to Tris-HCl.
Resolve different thiol proteins by sequential elution: 5–25 mM L-cysteine < 0.05 M glutathione
< 0.02–0.05 M 2-mercaptoethanol < and 0.02–0.05 M dithiothreitol in 50 mM Tris-
HCl, 1 mM EDTA, pH 7–8.
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