V '
C ells incubated w itli primary' antibody w here they w ill bind any antigen present. Y = antibody
3. Incubation with secondary antibody.
Secondary antibody conjugated w ith a fluorophore (*) applied.
I
Figure 2.15 Overv iew o f the procedure for staining ce lls for im m u nofluorescence m icroscopy.
2.14 Radioligand binding.
Procedure fo r tritiated-glibenclamide binding assay to cell homogenates.
Stable cell lines were grown to 100% confluence in a 500cm^ tissue culture dish as described in previous sections (see Sections 2.2, 2.11). Cells were washed twice with 25ml ice-cold binding buffer (50mM TrisHCl, pH7.4) before harvest in 10ml binding buffer using a cell scraper. Cells were aliquoted into 1ml aliquots and stored at -80°C until required. Cells were thawed at room temperature just before use and centrifuged at 10,000g for 3 minutes at 4°C to pellet the cells. The supernatant was removed and the cell pellet resuspended in 1ml binding buffer containing protease inhibitor cocktail and homogenised in a tight-fitting glass-on-glass dounce homogeniser (see Section 2.11). A small aliquot o f homogenate (20pl) was used in an assay to determine protein concentration (Bio-Rad protein assay kit) to enable specific binding activities to be calculated. A fraction o f the homogenate was used directly in each binding reaction (corresponding to 60fxg o f protein). Binding reactions were done in the presence o f 0-20nM ^H-glibenclamide (37MBq/ml, DuPont NEN, Brussels, Belgium) to assess specific binding. Non-specific binding was determined by parallel incubations in the presence o f 2pM unlabelled glibenclamide added from a
lOmM (w/v) stock in dimethylsulphoxide (Sigma, Poole, UK). The final reaction volume was 750pl. Binding reactions were allowed to come to equilibrium for 2 hours at room temperature with gentle rotation before the reaction was stopped by the addition o f 1ml ice-cold binding buffer. Immediately after the reaction was stopped bound ligand was separated from unbound ligand by vacuum filtration through Whatman GF/B filters. Filters were washed four times with 2ml ice-cold binding buffer before radioactivity bound to the filters was determined by liquid scintillation counting in 10ml Ultima Gold MV scintillation fluid (Packard, Groningen,
Netherlands) using a Beckman LS6000TA liquid scintillation counter. Each binding reaction was counted in triplicate, that is, each binding reaction was separated into three equal aliquots before each was stopped and filtered individually.
Ligand depletion was estimated empirically using a centrifugation-binding assay (Hulme 1992). A binding reaction was set up and incubated in the manner
described above. The bound radioactivity was separated from the unbound by centrifugation at 10,000g for 3 minutes at 4°C. The radioactivity in the supernatant and pellet was measured by liquid scintillation counting as described above. The centrifugation assay was done at the lowest concentration point (where ligand depletion will have most effect) and only those binding curves where depletion was less than 2 0% were selected for analysis.
Analysis o f binding data.
The raw data obtained from liquid scintillation counting expressed
radioactivity bound to filters in counts per minute (c.p.m.) rather than disintegrations per minute (d.p.m.). Data expressed in counts per minute are not corrected for the inefficiency o f the counter in detecting individual disintegration events. D.p.m. can be defined as the number of atoms o f a radioactive substance decaying, resulting in the emission o f ionising radiation, per minute. The detection efficiency o f the counter should be calculated by calibration against a standard o f known activity (1 becquerel is defined as 1 disintegration per second). Once the detection efficiency o f the counter is known it can be used to convert counts per minute into disintegrations per minute. It is preferable to express raw binding data in disintegrations per minute as it gives a direct measure o f the amount of bound radioactivity (assuming the specific activity of the radioactive ligand is known). However, in my binding experiments I was unable to calibrate the scintillation counter due to the absence o f appropriate standards for tritium. As a result o f this practical difficulty, counts per minute were converted to ^H- glibenclamide bound using a calibration curve constructed from a plot of amount of glibenclamide against counts per minute. The ^H-glibenclamide (of known specific activity) was added directly to scintillant and counted in triplicate as described above. The calibration curve was repeated for every new batch o f ^H-glibenclamide used. The points on the curve were fitted by linear regression using the Graphpad Inplot (v3.1) charting package.
For each binding experiment two sets of data were obtained corresponding to total binding and non-specific binding. As mentioned above all concentration points on the curves were done in triplicate. For the non-specific binding curve the mean
values were plotted and fitted by linear regression using the Graphpad software. The values of non-specific binding at each concentration point were taken by interpolation fi"om this line to obtain the “correction factor”. The triplicate values obtained at each concentration point for total binding were each converted to specific values by subtraction o f the appropriate “correction factor”. These corrected data values were fitted to the following equation;
y= Vmax. x
(Kd + x)
where Kd is the binding activity, Vmax is the maximal amount o f glibenclamide bound, y is the amount o f glibenclamide bound and x is the concentration of glibenclamide in the binding reaction. Data fitting was done using the Graphpad Inplot software package.
The Hill co-efficient o f binding was calculated from the gradient o f a plot of log(P/l-P) against log(ligand concentration), where P is the fractional occupancy. The fi*actional occupancy was calculated by dividing the mean o f the specific counts per minute at a given concentration point by the maximal occupancy (Vmax), as