Protein Protocols Protein Protocols

304 Jenö and Horst
BT1 membrane, or microleaks in the trap. In such cases, we prefer to run several preparative
gels in parallel and pool multiple protein slices until the required amount of protein
for electroelution is obtained. For low amounts of protein, several commercially available
devices can be used which elute proteins into smaller volumes than the BIOTRAP apparatus.
4. Electroelution of proteins into ammonium hydrogen carbonate would be preferable, since
NH 4HCO 3 can be removed by lyophilization. However, owing to the low buffering
capacity of NH 4HCO 3, the pH of the buffer drops after 4 h, rendering electroelution of
high molecular weight proteins difficult due to their slow elution from the gel pieces.
Therefore, it is preferable to electroelute proteins into Tris–glycine–SDS followed by
electrodialysis into NH 4HCO 3-containing buffers.
5. When using 0.1% SDS in the electroelution buffer, the micelles formed in front of the
BT1 membrane lead to massive accumulation of SDS in the trap. The detergent and low
molecular weight contaminants can be easily removed by hydrophilic interaction
chromatography with simultaneous desalting of the protein into a volatile buffer (7). Alternatively,
the procedure devised by Simpson et al. (6) can be used to desalt the
electroeluted protein.
6. Hydrophilic interaction chromatography of proteins requires careful control of the solvent
composition. To test column performance, we use a test mixture consisting of cytochrome
c, ovalbumin, and bovine serum albumin. Use of this test mixture allows finding the minimal
n-propanol concentration at which the electroeluted protein binds to the stationary
phase without the risk of precipitation by the organic solvent. Protein binding usually
occurs at n-propanol concentrations between 60% and 65%. When performing electroelution
with Tris–glycine buffers, direct application of the electroeluate is not possible due to
precipitation of buffer salts above 50% n-propanol concentration. In such cases, buffer
exchange into 0.1 M NH 4HCO 3, 0.01% SDS is carried out by electrodialysis in the
electroelution apparatus. Ammonium bicarbonate can be removed by Speed Vac drying.
The residual SDS facilitates solubilization of the dried protein with water. No salt precipitation
is observed when adding n-propanol to 65% final concentration
The solvent system used to elute bound proteins contains 50 mM formic acid. Unfortunately,
this precludes detection of proteins at wavelengths below 250 nm. Since most
proteins contain tyrosine and tryptophane residues, detection of eluting proteins is done at
280 nm. For proteins lacking tyrosines or tryptophanes, 0.05% trifluoroacetic acid (TFA)
can be used instead of formic acid. However, exposure of the stationary phase to TFA
should be kept to an absolute minimum, since TFA greatly reduces the lifetime of the
column.
Efficient removal of small molecular weight contaminants such as SDS and Coomassie Blue
requires small injection volumes of the electroeluted protein. When using 4.6 mm i.d.
columns, 50 µL injections were found to be optimal; for 2.1 mm i.d. columns, the injection
volume is reduced to 20 µL. Larger volumes are concentrated on the column inlet with
multiple injections. In such cases, the column should be allowed to reequilibrate between
individual injections, otherwise loss of protein in the breakthrough volume occurs.
Electroeluted proteins chromatographed by hydrophilic interaction often display
unsymmetrical peaks. This may indicate the presence of several different proteins in the
eluate, which are partially resolved by the stationary phase, or heterogeneity of a single
protein, which became modified during electrophoresis. Since the main purpose is to
free the electroeluted protein from SDS and Coomassie Blue, we tend to elute the protein
into one single peak by running very steep gradients, rather than trying to separate
the eluting material into single components. This can be subsequently achieved by
two-dimensional gel electrophoresis, or by reverse-phase chromatography after removal
of residual n-propanol.