Taken from booklet published by Pharmacia

CNB4-activated Sepharose 4B is produced by the reaction of Sepharose 4B, a bead-formed agarose gel, with cyanogen bromide. The active product is freeze-dried in the presence of dextran and lactose. Free cyanogen bromide is absent. 1 g freeze-dried material is equivalent to approximately 3.5 ml sedimented gel.

CNBr-activated Sepharose 4B is supplied in air-tight packs of 15 g and should be stored in a refrigerator below 8° C. Under these conditions the shelf life is approximately 18 months. The opened package must be stored dry below 8° C.

Sepharose 4B as a matrix

The nature of the matrix to which the proteins are attached is important in several respects. It must be physically and chemically stable under the experimental conditions, and columns packed with the matrix must have satisfactory flow properties. The use of Sepharose 4B in gel filtration and its suitability as a chromatographic medium are well documented. Since the use of immobilized proteins depends on specific adsorption, the matrix must be free from non-specific adsorption effects which could obscure the desired separation or interfere with the action of the enzyme. Effects due to charged groups as in copolymers of malaic anhydride and ethylene or to "unspecific factors" as in porous glass are minimal for Sepharose even for very large molecules. The open pore structure of Sepharose 4B, which allows it to be used for gel filtration of proteins up to 20 million daltons, enables large molecules to be coupled in good yield and ensures that their binding activities will be available even for large molecules. The closed pore structure of cellulose and conventional polyacrylamide gels is distinctly disadvantageous. Sepharose 4B displays virtually all the desirable features of a matrix for the immobilization of proteins and is thus the matrix of choice for the preparation of bed materials for affinity chromatography, and for the preparation of immobilized enzymes.

A large number of reaction schemes has been proposed for the coupling of proteins and other biopolymers to insoluble matrices, and the cyanogen bromide method results in an active product to which substances may be covalently coupled easily under mild conditions. Cyanogen bromide reacts with the hydroxyl groups of Sepharose to form imidocarbonate and carbamate groups.

During the subsequent coupling of a protein to the active product, the imidocarbonate groups react with amino groups belonging to the protein with the formation of stable covalent linkages. On the basis of studies of model reactions with methyl 4, 6-O-benzyline- a -D-glucopyranoside, it seems reasonable to conclude that the main part of the product is coupled through isourea derivatives.

Swelling and washing the gel

Dextran and lactose, which are added to the activated gel to preserve its activity under freeze-drying, are washed away during the swelling stage. The required amount of gel is swollen in 10^-3 M HC1 solution on a glass filter and washed for 15 min. with the same solution. The protein-binding activity of the gel is preserved better by washing at low pH than by washing at pH's above 7. Note that 1 g of freeze-dried material gives a final gel volume of approximately 3.5 ml. Approximately 200 ml solution is added in several aliquots for each gram of dry gel, the supernatant being sucked off between successive additions. Immediately after washing, a solution of the protein to be coupled is added.

Since the optimal conditions, e.g. pH, buffer composition, temperature, for the coupling of a particular protein will depend to some degree on its properties, it is not possible to give an exactly specified procedure which will give optimal results in every case. It is, however, possible to give a general indication of the conditions which are likely to prove most effective.

The coupling reaction proceeds most efficiently in the pH range 8-10, but a lower pH may be used if the stability of the protein requires it. The protein to be coupled should be dissolved in a buffer solution of high ionic strength, about 0.5, to reduce protein-on-protein adsorption caused by the polyelectrolyte nature of proteins, and to facilitate the subsequent washing procedure. Carbonate/bicarbonate and borate buffer systems with the addition of NaCl may be used. The coupling reaction may be conveniently followed by observing the decrease in the absorbance of the supernatent solution e.g. at 280 nm, but this will generally give an overestimate of the amount of protein covalently bound.

The amount of protein which couples under a given set of conditions depends mainly on the ratio of protein to gel volume, the pH of the reaction and the protein itself as well as the duration and temperature of the reaction.

After coupling is complete, the excess protein is washed away with coupling buffer solution on a glass filter, and any remaining active groups are blocked by treatment with 1 M ethonolamine at pH 8 or 0.2 m glycine for 2 hours. The final product is then washed alternately with high and low pH buffer solutions four or five times. Acetate buffer solution (0.1 M, pH 4) and borate buffer solution (0.1 M, pH 8.5), each with the addition of 1 M NaCl, or equivalent buffer systems may be used.

Thorough washing of the coupled product is necessary to remove traces of non-covalently adsorbed materials which might otherwise disturb subsequent experiments and the washing-cycle of low and high pH recommended above is essential for the best results. Protein desorption only occurs when the pH is changed. After three or four washing cycles, the amounts of protein desorbed are very small, and decrease rapidly with further pH changes. It should be noted that pH changes do not cause loss of protein which is covalently bound to the Sepharose matrix, and that even physically adsorbed protein does not desorb unless the pH is changed.

Protein coupled to CNBr-activated Sepharose 4B is usually more stable to denaturation than the protein in free solution, but reasonable care in the choice of storage conditions should be exercised. Suspensions should be stored in a refrigerator below 8° C in the presence of a suitable bacteriostatic agent. The choice of buffer solution depends on the properties of the particular coupled protein.

1. Prepare Sepharose immunoadsorbant as described above.
2. Stir Sepharose and quickly add protein solution.
3. Check pH. It should be pH 9.0. If not, adjust it.
4. Maintain pH at 9.0 for 1 to 2 hours at room temperature.
5. Make 1M in ethanolamine pH8 or 0.2M glycine for 2 hr - overnight
6. Wash the column as follows

1. 5 volumes, BSB (borate saline buffer pH 8.6)
2. 1 volume, 1M ethanolamine or 0.2M glycine, pH 8.2
3. 5 volumes, BSB

COLUMN IS READY TO USE

1. Pass serum or other solution thru the column slowly. Flowrate should be about 1 ml/min. Then wash column with BSB until O.D. in effluent is low.
2. To elute protein, wash top of column with small amounts of buffers below in order to wash protein into the column and not up into the buffer on top of the column. Use 1 ml at a time, but try to avoid letting the column go dry.
3. In order to elute protein from column, use:

Just before the pH of the column changes, the eluted protein is rapidly coming off. Collect protein in test tubes containing BSB or 1M Tris pH 7.5 so that the solution is rapidly neutralized or at least that the pH is raised enough to avoid too much denaturation of protein. Read O.D.'s of tubes to locate the protein eluted. After the protein has been eluted from the column, begin dialyzing it immediately against BSB. Also, quickly re-equilibrate column with BSB to avoid denaturation of proteins linked to it.

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