Thiopropyl Sepharose® 6B
Thiopropyl Sepharose® 6B contains reactive 2-thiopyridyl disulphide groups attached to Sepharose through a chemically stable ether linkage, see ﬁg. 1.
Fig. 1 . Partial structure of Thiopropyl Sepharose 6B.
Thiopropyl Sepharose 6B reacts with solutes containing thiol groups under mild conditionsto form mixed disulphides. This reaction forms the basis of covalent chromatography and a procedure for reversible immobilization of thiol containing molecules. These techniques make it possible to: • separate thiol-containing proteins and peptides from non-thiolcontaining proteins and peptides • in certain cases separate enzymes with active-site thiol groups from denatured enzymes • store andprotect thiol-containing proteins • regenerate both the coupled biomolecule and the active bed material Because it is less porous than Activated Thiol Sepharose 4B, Thiopropyl Sepharose 6B is suitable for immobilizing low molecular weight substances. For immobilizing high molecular weight substances, we recommend Activated Thiol Sepharose 4B from Amersham Pharmacia Biotech. 71-7105-00
Edition ABTable 1. Gel characteristics.
Active group: Active group concentration: Spacer: Coupling capacity: 2-pyridyl disulphide
18–31 µmole 2-pyridyl disulphide/ml drained gel 2-hydoxypropanol approx 10–20 mg protein/ml drained gel, e.g. 14 mg ceruloplasmin (MW 132 000) /ml drained gel Bead structure: 6% agarose Bead size range: 45–165 µm Mean bead size: 90 µm Max linear ﬂow rate*: 75 cm/h at 25 °C,HR 16/10 column, 5 cm bed height pH stability** Long term: 2–8 Cleaning-in-place: 2–8 Chemical stability: Stable to all commonly used aqueous buffers and additives like detergents. Azides should be avoided. Physical stability: Negligible volume variation due to changes in pH or ionic strength. Autoclavable: Not recommended
Linear ﬂow rate= volumetric ﬂow rate (cm3/h) column cross-sectional area(cm2)
** The ranges given are estimates based on our knowledge and experience. Data refer to the coupled product, provided that the ligand can withstand the pH or chemical environment. Please note the following: pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pHstability, short term refers to the pH interval for regeneration and cleaning procedures.
Covalent chromatography differs from other types of chromatography in that covalent bonds are formed between the gel and molecules in the mobile phase. Thiopropyl Sepharose 6B reacts with solutes containing
thiol groups to form a mixed disulphide and release 2-thiopyridone, see Fig.2. The solute is thus covalently linked to the gel from which it can be subsequently eluted by addition of a reducing agent, e.g. 2-mercaptoethanol, DTT or glutathione, see Fig. 2. Note that additional oxidizing reagents are not used and the risk of disulphide formation between molecules in solution is minimized.
R+S N H
SH + RSH + R'S
Fig. 2. Reaction scheme for covalent chromatography of a thiolated substance (RSH) on Thiopropyl Sepharose 6B. R'SH represents a low molecular weight thiol such as dithiothreitol.
Preparing the gel for covalent chromatography
Thiopropyl Sepharose 6B is supplied freeze-dried in the presence of additives, which must be washed away at neutral pH before coupling. Distilled wateris recommended for swelling and washing. Weigh out the required amount of freeze dried powder (1 g freezedried powder gives about 3 ml ﬁnal volume of gel) and suspend it in distilled water. The gel swells immediately and should be washed for 15 minutes with distilled water on a sintered glass ﬁlter. Use approximately 200 ml per gram freeze-dried powder, added in several aliquots. Prepare a slurry...
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