To overcome sample interactions with charged sulfate groups on agarose resins and charged carboxyl groups on dextrans, a minimum salt concen-tration of 0.15 M should be used in eluting buffers. The opposite may be the case when using acrylamide-based resins as some proteins in the presence of high salt may show hydrophobic interactions. All eluting buffers should be set at a pH chosen to maintain the stability of the target protein and to maintain the integrity of the resin (Table 5.1). If the sample is to be concentrated by freeze drying after the chromatographic run, a volatile
buffer (with no salt addition) should be used as the mobile phase (Table 2.8). Any mobile phase buffer should also be degassed and filtered through 0.2 µm membranes before use in SEC.
5.3 Preparation and storage of SEC resins
SEC resins are supplied either preswollen in 20% (v/v) ethanol or as a dry powder. To use the preswollen resins the preservative can be removed by filtration, the resin can then be resuspended in the desired buffer and degassed under vacuum before being packed into a column (see Section 1.11). The dry powders can be swollen for use in an excess of buffer overnight or heated in a water bath for 3 h at 100°C. After allowing the gel to cool and settle, any floating material (‘fines’) should be poured off prior to packing into a column. If not removed these ‘fines’ (fragments of resin) will lodge in the pores of the beads and will eventually cause problems.
When an SEC column has been packed and calibrated it can be left (with the inlet and outlet tubes blocked off) in a storage area after two column volumes of preservative (see manufacturer’s recommendations) have been pumped through the resin.
5.4 Analytical SEC
To assess the column-packing efficiency and to determine the V0 and Vt
of the column, a solution of blue dextran (Mr 2 000 000) and vitamin B12
(pink; Mr 1385) or potassium dichromate (yellow; Mr 294) can be used.
The progress of the colored compounds down the column can be monitored and these should flow down the column in even, level bands.
Any deviation, such as tailing or uneven distribution, will indicate uneven packing which will result in flow distortion of applied sample and may require the column to be repacked.
The elution volume of blue dextran and vitamin B12 (or potassium dichromate) is from the midpoint of the sample volume application to the midpoint of the elution volume (Figure 5.3). If the packing is judged to be workable the column can be calibrated by the application of a series of standard proteins of known Mr, the elution volumes (Ve) of these proteins can be measured, their Kav values determined and used to construct a calibration graph for the column. The Kav of the standard proteins is plotted against the log10 Mr. A protein sample can be applied to the column, the Kav for the target protein can be measured and using the calibration graph an estimate of the target protein’s Mrin its native confor-mation can be determined (see Exercises 5.1 and 5.2).
5.5 SEC to separate protein aggregates or the removal of low amounts of contaminating material
If, after trying separations based upon charge (see Sections 3.1–3.3 and 6.5), biospecificity (see Section 4.1) and hydrophobicity (see Section 3.4) there are still low levels of contaminating protein, separation based upon size using SEC may be appropriate. The chromatographic run using an SEC
resin will have the dual benefit of separating the target protein from other proteins with a different size and shape as well as any dimers and oligomers of the target protein. The disadvantage is that SEC will dilute the target protein.
5.6 Desalting (group separation)
At different stages in a purification protocol a change of buffer may be required prior to application to another resin or prior to electrophoresis. The exchange of buffer ions can be achieved by dialysis (see Protocol 2.9), but the process is time consuming. An alternative to dialysis can be achieved with SEC using resins with a small pore size which totally exclude the proteins in the sample applied and totally include the buffer ions.
Sephadex G-25 or G-50 (GE Healthcare) and Biogels P-6 or P-10 (Bio-Rad) resins can be used for desalting (or prepacked columns of G-50 (PD-10) can be purchased (GE Healthcare). Preswollen resin can be packed into a syringe with glass wool at the base (to prevent the resin leaving the column) and equilibrated with the buffer to be exchanged into. The sample in the buffer that is no longer required is applied to the resin (sample:SEC resin volume ratio should be 1:5) and fractions are collected.
The protein will elute early in the new buffer because it is excluded from the gel; the buffer ions that are not required will elute later. Desalting requires significantly less time than dialysis and is favored in industry for buffer exchange.
5.7 SEC in the refolding of denatured proteins
Prokaryotic protein expression systems will not have the necessary chaper-ones to correctly fold eukaryotic proteins into their tertiary structure (see Section 4.7). Proteins that have been expressed in the bacteria Escherichia coli may form cytoplasmic aggregates called inclusion bodies. The extrac-tion of these expressed recombinant proteins will require the use of chaotropic salts (e.g. urea) and detergents (e.g. SDS). After extraction the solubilizing agents will have to be slowly removed to allow the proteins to fold into their correct conformation. The reduced diffusion in SEC has been used to promote the correct refolding of proteins by suppressing nonspecific protein–protein interactions. In addition, during SEC the aggregates and solubilizing buffers are removed in a single experiment.
5.8 Preparative polyacrylamide gel electrophoresis (PAGE)
Polyacrylamide gel electrophoresis is a highly resolving technique which is routinely used to monitor the purity of pooled fractions produced by chromatographic separations (see Section 6.1). Analytical electrophoresis can be viewed as incomplete electrolysis because the experiment is stopped before the sample reaches the electrode and the samples within the gel are visualized by staining. If a PAGE experiment is allowed to proceed (after the bromophenol blue tracking dye has run off the end of the gel) the applied sample will eventually emerge from the bottom of an analyt-ical gel and enter the anodic buffer chamber.
This is analogous to a chromatographic separation and there are a number of proprietary apparatuses available for the purification of proteins using either nondenaturing or denaturing preparative polyacrylamide gel electrophoresis (see Section 6.3). In these systems the sample is applied to a column of acrylamide and run using conventional PAGE buffer systems.
The end of the acrylamide column rests upon a sintered glass disc (Figure 5.5) through which a buffer flows tangentially to the column, removing protein and/or polypeptides as they emerge from the bottom of the acrylamide gel, taking them to a fraction collector. Proteins are prevented from contacting an electrode by the use of dialysis tubing which also maintains the electrical integrity of the system.
Preparative gel apparatus rarely gives excellent resolution of bands that appear close together on an analytical gel. This is because as the bands emerge from the bottom of the acrylamide column, mixing takes place in the eluting buffer (reducing the amount of sample applied and reducing the dead volume between the preparative PAGE apparatus and the fraction collector can help reduce sample mixing). For these reasons, it is not an ideal technique to use in the early stages of a purification protocol when the target protein is usually a small percentage of the total protein.
However, it can be used successfully in the later stages of a purification protocol to remove trace contaminants. If the temperature is controlled during a nondenaturing preparative PAGE experiment (separation based upon surface charge and relative mass; see Section 6.3) the sample will
Cathode –ve
Sintered glass
Elution Pump buffer
Anode +ve
Upper buffer chamber
Detector and fraction collector
Rubber bands
Dialysis tubing
Lower buffer chamber Stacking gel
Resolving gel
Figure 5.5
A typical preparative polyacrylamide gel electrophoresis set-up.
emerge from the experiment in an active conformation. If the purity of the sample is still not acceptable the active fractions can be concentrated (see Section 2.14), incubated with SDS and 2-ME and rerun on the same polyacrylamide column (the electrode buffers would require changing for denaturing PAGE) isolating the sample this time on molecular mass.
If the target protein can be identified on an analytical gel using (i) an antibody to the target protein (western blotting; see Protocol 6.3), (ii) detecting the protein’s activity in situ (nondenaturing PAGE; see Section 6.3), or (iii) using the Mr of the target protein (denaturing PAGE; see Section 6.3), the band containing the target protein can be excised from the gel. Preparative well-forming combs coupled with wider gel spacers (0.75, 1.0 and 1.5 mm) will enable larger amounts of the target protein to be applied. However, there is an upper limit to the amount of protein that can be applied to any acrylamide gel which depends on the number and relative amounts of proteins in the sample. Localized heat spots can be generated at high protein loadings as the protein concentrates within the gel during the electrophorectic run, resulting in band distortion.
5.9 Isolation of proteins from polyacrylamide gels or from nitrocellulose membranes
Electroelution
Proprietary electroeluting apparatus can be used which moves the sample by electrophoresis from the gel into a collection chamber. The sample is prevented from contacting the anode by dialysis membrane. Alternatively, gel slices can be placed into a dialysis bag in buffer and then draped over a horizontal electrophoresis apparatus. Minimum buffer can be used in the dialysis bag to contact the two electrodes. The proteins within the gel will migrate into the buffer, where they can be collected. While the electrical field is in place they will continue to move towards the anode. Over time this can result in proteins contacting the dialysis membrane; to overcome this at the end of the electroelution the current polarity should be reversed for 2 min to propel the proteins back into the buffer. Stained and unstained proteins can be electroeluted and the recovery of protein is usually higher when SDS is included in the electrode buffers.