Biomedical Engineering Reference
In-Depth Information
Fig. 1. Schematic of the layout for a 10-point saturation radioligand binding
experiment.
5. Terminate reaction by rapid fi ltration over Whatman GF/B fi lters using a cell
harvester. Wash fi lters three times with 3 mL of ice-cold wash buffer.
6. Place fi lters in scintillation vials, add cocktail, and count in a scintillation counter
(
see
Note 12
).
7. Determine protein concentration of membrane homogenate.
3.1.3. Analysis of Radioligand Binding Data (
see
Note 13
)
The fi rst step in the analysis of binding data is to determine the mean of
the duplicate values obtained from the scintillation counter. If the scintillation
counter expresses these data as counts per minute (cpm), convert the data to
disintegrations per minute (dpm) using the following equation:
cpm/counter effi ciency for [
3
H] = dpm
The next step is to determine the exact concentration of each concentration
of radioligand used using the following equation:
dpm
———————————————————————————— = Conc. (
M
)
2.22e12 dpm/Ci
×
specifi c activity (Ci/mmol)
×
volume counted (mL)
Search WWH ::
Custom Search