Biomedical Engineering Reference
In-Depth Information
and dry the pellet with air. After dissolving the pellet in an alkaline
solvent, the sample may be used for protein determination.
References
Wessel D, Flügge UI (1983) Anal Biochem 138:141
Polyethylene glycol (PEG) is well suited for gentle concentration of
proteins. Among the PEGs of different degree of polymerization,
the most effective is PEG 6000. The concentration range of PEG
6000 is between 0 and 15% (w/v) for fractionation. As examples,
circulating immune complexes are precipitated with 3.5% PEG 6000
(w/v, final concentration) in 0.1 M borate buffer, pH 8.4; membrane
protein complexes are precipitated at 0
◦
C with 7.5% PEG 6000 (w/v,
final concentration) in 50 mm Tr i s , 1 0 mmmgCl
2
,pH7.4.
Complete precipitation by PEG needs 30 min to 15 h at 0
◦
C.The
precipitate is collected by centrifugation or filtration on glass fiber
filters (e.g., Whatman GF/A). Dissolve the precipitate in ddH
2
O
and dialyze against an appropriate buffer. An increase in volume
occurs during dialysis.
3.7.4 Lyophilization (Freeze Drying)
During lyophilization the water of deeply frozen samples is re-
moved by sublimation in vacuum. The structure of a probe is well
conserved, especially if additives as sucrose or trehalose are added.
In a sealed bottle, the sample can be stored at RT for years. If the
buffer constituents are to be removed also, the sample has to be
dissolved in a volatile buffer (see Table 7.6). Reconstitute the sample
by addition of water or, in the case of volatile buffers, by addition
of a buffer of your choice.
For optimal conditions it is necessary to freeze the sample very
quickly in a methanol-dry ice bath or in liquid nitrogen and to
store the frozen sample below −30
◦
C until the vacuum of less than
100 Pa (0.0145 psi) is reached and the lyophilization is finished.
During the lyophilizing process, the sample has to get external
heat because sublimation of ice withdraws heat resulting in cooling
the probe, but avoid melting of the sample.
Concentration of a sample in a SpeedVac, a centrifuge with
open rotor running in vacuum, is not lyophilization because the
sample is not frozen; therefore, this method does not fit for macro-
molecules with defined structures. The reconstitution (dissolving)
of protein samples concentrated in a SpeedVac is often more dif-
ficult and comparable to drying with air. But it is the method of
choice for concentration of solutions of low-molar mass substances,
e.g., peptides, since the sample is collected at the bottom of the tube
by means of centrifugal force.
