Biomedical Engineering Reference
In-Depth Information
to drain briefly (<20 sec), but not by squeezing as had been proposed by others (Zahn et al.,
1973). Intact tubules prior to expulsion were obtained by dissection of animals that had been
euthanised by freezing at minus 20 °C.
2.3 Microscopy
To look for the presence of glycoprotein on the surface of expelled tubules, samples were
treated with fluorescently-labelled lectins; fluorescein isothiocyanate (FITC)-labelled
concanavilin A (ConA), FITC-labelled
Datura stramonium
agglutinin (DSA), and FITC-
labelled
Lycopersicon esculentum
agglutinin (LEA) (all from Sigma, St Louis). All FITC-
labelled lectins were applied as 20 μg/mL solutions in Tris-buffered saline (TBS) for 60 min,
followed by 3 × 5 min washes in TBS. Samples were examined using appropriate narrow
pass filters on an Olympus BX61 fluorescence microscope.
To examine the distribution of adhesiveness on tubules, individual freshly expelled tubules
after draining (see above) were transferred to a wash solution in a plastic trough which
contained a suspension of 0.5% w/v Bio-Gel P2 (45-90 m particle size) in 3.5% w/v NaCl,
10 mM sodium phosphate, pH 7.6. After 5 sec immersion, the tubules were washed 3 times
in 3.5% NaCl, 10 mM sodium phosphate, pH 7.6 and were then drained and placed onto
glass slides. After air drying, the tubules were examined by microscopy.
For scanning electron microscopy (SEM) expelled tubules were examined using a Philips
XL30 FESEM microscope at an accelerating voltage of 2 kV.
2.4 Gel electrophoresis analysis
Freshly expelled and drained tubules were allowed to adhere to a glass plate and were air
dried. The tubules on glass plates were removed by peeling, leaving the layer of adhesive,
and potentially other components of the tubule wall as a print on the glass (DeMoor et al.,
2003). This material was collected by removal with a sharp razor blade and was then
extracted in electrophoresis sample buffer, containing 2-mercaptoethanol. SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) was based on the method of Laemmli
(1970) using Invitrogen NuPAGE Novex 4-14% Bis-Tris Gel with MES running gel buffer, at
180V for 60 min. Molecular weights were determined by comparison to globular protein
standards (BioRad) using BioRad Quantity One v.4.4.0 software. For protein identification,
gels were stained by Coomassie Blue R-250. Samples that had not been dried completely,
but only sufficient to remove excess liquid, appeared to give samples that contained less
insoluble material, although the yield of adhesive proteins was less.
2.5 Amino acid analysis
Protein extracts were separated by SDS-PAGE, followed by transfer of the protein bands to
PVDF membrane using Invitrogen NuPAGE Transfer buffer (NP0006-1). Amino acid
analysis of PVDF membrane pieces used vapour-phase hydrolysis (5.8 M HCl at 108 °C for
18 h), followed by precolumn derivatisation with 6-aminoquinolyl-
N
-hydroxysuccinimidyl
carbamate (Cohen & DeAntonis, 1994). Derivatives were separated and quantified by
reversed phase (C18 Waters AccQTag) HPLC at 37 C (Cohen, 2001) (Australian Proteome
Analysis Facility), using a Waters Alliance 2695 Separation Module, a Waters 474
Fluorescence Detector and a Waters 2487 Dual Absorbance Detector in series.
