Protein Protocols Protein Protocols

302 Jenö and Horst
Fig. 1. Side (A) and top (B) view of the electroelution device. The apparatus is assembled
by inserting a BT1 membrane at points A and G. The trap that collects the protein during the
elution process is formed between points F and G. The chamber that holds the gel pieces is
formed by inserting a BT2 membrane at point C. Smaller elution chambers can be made by
inserting the BT2 at point D or E. The trap inserts and membranes are fixed by clamping plates,
which press the trap inserts against the cell body. 1, Cell body; 2, pressure screw; 3, clamping
plate; 4, trap inserts; 5, membrane BT1; 6, membrane BT2; 7, trap chamber; 8, elution chamber;
10, mark for correct orientation of membrane BT1; 11, trap insert for membranes BT1
(modified with permission from Schleicher and Schuell).
6. Dissolve the dried protein in a small volume of water (50–100 µL). The dried SDS efficiently
solubilizes the protein. n-propanol is added to 65% final concentration. The sample
is then applied in 50-µL aliquots onto the PHA column. After each injection, a number of
UV-absorbing peaks, caused by Coomassie blue components, elute from the column. It is
important that these components are completely washed out before the next aliquot is
injected. With this procedure, the protein is efficiently concentrated on the column inlet.
After the entire sample has been applied, the gradient is initiated, which is developed in
10 min from 65% n-propanol/50 mM formic acid to 50 mM formic acid. The protein
elutes at the end of the gradient and is now devoid of any salt or SDS (Fig. 2).
7. The protein is now ready for further protein structural characterization. It can be directly
subjected to automated Edman degradation. For enzymatic fragmentation, residual n-propanol
has to be removed in the Speed Vac prior to adding the protease.
4. Notes
1. To locate the protein of interest in the gel, a staining method has to be chosen so that
maximal sensitivity with minimal fixation is obtained. A number of methods exist to
visualize proteins in the polyacrylamide matrix, such as formation of insoluble