Biomedical Engineering Reference
In-Depth Information
3.2 Gel electrophoresis analysis
The adhesion print isolated from glass showed a range of proteins of well-defined molecular
weights when analysed under reducing conditions by SDS-PAGE (Figure 4). A similar
pattern, except as noted below, was observed from >10 separate samples from different
animals. Seven bands, H2 at 89 kDa, H3 at 70 kDa, H4 at 61 kDa, H6 at 44 kDa, H7 at 37
kDa, H8 26 kDa and H9 at 17 kDa, were consistently present in all tubule samples that were
examined (n > 12). Band H4 has been examined as a single entity, but in some gels (Figure 4)
it appeared that it may comprise 2 components. For all other bands, although each appeared
to be a single component, it is also possible that more than one component could be present,
migrating similarly. In many samples, but not all, an additional band, H5 at 53 kDa, was
present. As it was not consistently present it was assumed that it may not be a key
component of the adhesive system and hence was not examined further. In a few samples,
an additional band H1 was observed at 150-170 kDa. This band seemed to be more prevalent
in samples where tubule fragments were present in the adhesion print, and could possibly
be related to the collagen that is the main structural component of the tubule (Watson and
Silvester, 1959). The collected material sometimes contained a proportion of material that
remained insoluble in the sample buffer. Examination of the adhesive prints under a
microscope suggested that the samples that subsequently contained more insoluble material
also contained more fibrous material that could be from the collagenous wall of the tubule.
Samples with little wall material typically had little, if any insoluble material.
Fig. 4. SDS-PAGE of the reduced proteins from the surface of a freshly expelled Cuvierian
tubule from
H. dofleinii
. The gel was stained with Coomassie Blue R-250. Key bands are
labelled and their estimated molecular weights, interpolated from a standard curve using
globular protein standards (BioRad), are given.
