Basic column technique

B.1 Basic column technique

Figure B.1 shows a suitable basic arrangement of equipment for gel filtration.

B.1.1 Equipment

Columns. Many different types of column are available, each with their own advantages and dis-

advantages. Many manufacturers supply a wide range of apparatus, from simple manual columns to fully integrated systems.

Sample application

Chromatography column with gel

Peristaltic

pump

Valve Sample

UV analyser collector

Buffer reservoir

Fig. B.1 Equipment for column chromatography. The equipment shown allows efficient chromatographic separation of protein mixtures. It is an advantage to have an insulating jacket around the column to protect it from draughts and temperature changes. If the column is equipped with flow adaptors at either end, this will allow the column to be inverted and run in the opposite direction, or to be run with the buffer flow in the ascending direction which will lessen the chances of the column packing down.

A P P E N D I X B: Basic techniques and useful data 353

Pumps. Peristaltic pumps provide an even flow rate with little solvent turbulence. Alternatively, a simple reservoir such as a Marriotte flask can be used. It is important to maintain a constant pressure on the column.

Monitoring and collection of fractions. A fraction collector and flow-through UV analyser connected to a chart recorder are necessary.

B.1.2 Fractionation of serum on Sephacryl S-200

Sephacryl S-200 is a preswollen gel that excludes proteins of over 250 000 molecular weight and so is extremely useful for the isolation of IgM. The same principles apply to the use of other gels fractionating in different size ranges.

MATERIALS AND EQUIPMENT Sephacryl S-200 Phosphate-buffered saline (PBS) Column chromatography equipment (Fig. B.1) Serum

METHOD

1 De-gas the gel under a vacuum. Air bubbles in the gel will distort the protein bands during the run.

2 Pour the gel into the column along a glass rod to avoid air bubbles. Take great care that the column is vertical. All the gel must be poured into the column at one time. Use an extension tube or reservoir. Leave the column outlet open during packing. A column of 100 × 2.5 cm generally takes about 5 h to settle.

3 Once the gel has settled fit the flow adaptor.

4 Pack the column by running through at least 2 column volumes of buffer. The flow rate should be about 20 ml/h (faster flow rates are possible with Sephacryl). After packing lower the flow adaptor if necessary. If the column tends to pack down after several runs you are probably running the column too fast. If a flow adaptor is used, packing after extended use may be avoided by using descending and ascending flow chromatography alternatively.

5 When the column is not in use add thiomersal to a concentration of 0.005% to stop microbial growth. This must be completely flushed out of the column before adding a sample as it absorbs at the same wavelength as protein. Alternatively, columns may be run with buffer containing sodium azide 0.01 M . Azide gives less absorption at 280 nm.

Sample application

Gel surface must not be disturbed during sample application as this would cause distortion of the bands.

Feed the sample through the pump and then through the flow adaptor, but note, this will cause some mixing and dilution of the sample in the dead space of the pump tubing. If an adaptor is not available place a nylon net or a layer of G-25 Sephadex (about 5 mm) on top of the gel to protect the surface.

A P P E N D I X B: Basic techniques and useful data

Add a little sucrose to the sample to increase its density and layer it gently onto the gel surface below the free buffer with a long Pasteur pipette or a syringe with capillary tubing.

1 Apply a sample volume of up to 8 ml of serum.

2 Run the column at 20 ml/h.

3 Collect samples of 2–5 ml.

Distribution of serum proteins in eluted volume

Three major peaks should be eluted on fractionation of mouse serum. Peak 1

The first peak contains the macroglobulins, IgM and α 2 -macroglobulin, plus some lipoproteins. Also, on some occasions, large haemoglobin–haptoglobin complexes are present. These are easily distinguishable by their reddish-brown colour. Some IgA may be found towards the end of the elution of this peak.

Peak 2 Most of the IgG is in this peak, together with IgA in the first fractions.

Peak 3 This contains mainly albumin and other small globulins.

Occasionally the first two peaks are not resolved satisfactorily. This is due to weak non- covalent interactions between the IgG molecules causing them to aggregate and so contaminate the first peak. In these cases, run the column in 0.1 M acetate buffer, pH 5.0. Lowering the pH increases the positive charges on the IgG molecules increasing the repulsive forces between them, so preventing aggregation.

Examination of fractions

After concentrating the fractions, examine either with reference to their molecular weight, by SDS polyacrylamide gel electrophoresis (see below), or immunologically by immunoelectro- phoresis (see Chapter 3).

B.1.3 High-pressure liquid chromatography (HPLC)

Columns have been developed which have increased the HPLC molecular weight fractionation range sufficiently to deal with immunoglobulins. They are useful for analysing the products of enzymic digestion and for monitoring the purity of fractions from gel exclusion columns

(e.g. they are capable of resolving F(ab′) 2 from whole IgG) and they are very fast, taking typically

30 min instead of the hours needed on a conventional gel column.

Such columns are generally small with sensitive detection systems capable of detecting 1–5 µg of protein, making them more suitable for analytical than preparative purposes.

A P P E N D I X B: Basic techniques and useful data 355

B.1.4 Buffer exchange and desalting of protein solutions

Dialysis is often used to remove small molecular contaminants or to change the buffering con- ditions of a macromolecular solution. The same effect can be achieved rapidly by the use of a Sephadex G-25 column. Small molecules, e.g. free dinitrophenol, ions of the original buffer, are retarded by the Sephadex while protein molecules are excluded from the gel and can be collected in the effluent, already equilibrated in the column buffer.

Rapid dialysis is often important when the protein is under harsh conditions, where con- ventional dialysis would be too slow and would allow irreversible denaturation. The technique described is a similar principle to size fractionation of proteins by molecular sieving. But a word of caution: since the required molecules are excluded from the gel, they cannot be size fractionated, so filtration through the gel is faster and the whole procedure is less critical.

B.1.5 Preparation of exchange column

MATERIALS AND EQUIPMENT Sephadex G-25, fine

Chromatography column Blue dextran, 1% w/v in water Peristaltic pump for column

Determination of column void volume

1 Pour the Sephadex into a chromatography column and pack under pressure.

2 Ensure that the surface of the gel is level, open the flow-control valve and allow the water to completely enter the column. (With Sephadex G-25, the gel bead size is sufficient to support the column of water inside the gel by surface tension; consequently, liquid flow stops as the meniscus comes into contact with the top of the gel. With fine grades of Sephadex this does not happen. The column may dry out and crack if left to flow unattended.) Close the column outflow.

3 Add 1 ml of blue dextran solution to the surface of the gel. Allow this to enter the gel com- pletely while collecting the effluent into a graduated cylinder. Close the column.

4 Add water to the surface of the gel and continue collecting the effluent until the blue dye just appears. The liquid collected represents the void volume of the column.

Repeat the void volume determination for different heights of the gel. Plot a graph of void volume against column height. If you use the same diameter column each time then the void volume may be read off from the graph using the column height.

Molecules in the excluded fraction of Sephadex G-25, for example proteins, leave the gel just after the void volume, and their volume is expanded to approximately 1.5 times the original sam- ple volume.

B.1.6 Use of column for buffer exchange

MATERIALS AND EQUIPMENT Sephadex G-25 column 1% w/v blue dextran solution

A P P E N D I X B: Basic techniques and useful data

Peristaltic pump Protein solution to be dialysed

METHOD

1 Determine void volume of the column.

2 Equilibrate the gel with buffer; equivalent to 3 times the void volume of the column.

3 Apply the sample in a volume not greater than half the void volume.

4 Allow the void volume of buffer to leave the column and collect up to 1.5 times the original sample volume.

If you are using small sample volumes and mini-columns, for example during radioiodination of protein, it is advisable to determine the volume of the final sample more precisely by using a test volume of blue dextran equal to the original sample volume. This avoids unnecessary dilution.

After one run, the column may be re-equilibrated with 3 times the void volume of buffer, provided the retained material has not irreversibly bound to the column, e.g. as occurs with fluorescein isothiocyanate, or has not altered the gel chemically.