soaked in buffer and substrate. After an appropriate incubation period, the strips are
peeled apart and the paper zymogram treated accordingly to detect enzyme product;
hence, it is possible to identify the position of the enzyme activity on the original
strip. An alternative approach to detecting and semiquantifyinganyparticular protein
on a strip is to treat the strip as the equivalent of a protein blot and to probe for the
given protein using primary antibody and then enzyme-linked secondary antibody
(Section 10.3.8). Substrate colour development indicates the presence of the particular
protein and the amount of colour developed in a given time is a semiquantitative
measure of the amount of protein. Thus, for example, large numbers of serum samples
can be run on a wide sheet, the sheet probed using antibodies, and elevated levels of a
particular protein identified in certain samples by increased levels of colour develop-
ment in these samples.pl
4.0150[ ́ 103 ]
Mr
100
70
60
5040302015105.0 6.0 7.0Fig. 10.9A typical two-dimensional gel. The sample applied was 100mg of total protein extracted from
a normal dog heart ventricle. The first dimension was carried out using a pH 4–7 isoelectric focussing gel.
The second dimension was a 12% SDS–PAGE vertical slab gel. The pattern was visualised by silver staining.
(Courtesy of Monique Heinke and Dr Mike Dunn, Division of Cardiothoracic Surgery, Imperial College School
of Medicine, Heart Science Centre, Harefield, UK.)416 Electrophoretic techniques