Radioligand binding saturation curve protocol

Dopamine D47 (RBHD47M400UA) saturation curve
Assay buffer:
50 mM Tris-HCl pH 7.4, 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM
EDTA
Wash buffer:
50 mM Tris-HCl pH 7.4, 0.9 % NaCl
Radioligand
[3H]-MethylSpiperone
Cold ligand
Haloperidol
Unit size
35 µg protein / unit
Protein concentration
14 mg/ml
85.5 Ci/mmol
Full scale assay format
Total volume
Volume membranes
Volume cold ligand or buffer
Volume radioligand
550 μL
500
25
25
Membrane dilution
0.125ml membranes +
24.875 ml assay buffer
(1:200 dilution)
Haloperidol dilution
Stock concentration
Prepare 1000μL in assay
buffer
Final concentration
Dilution factor
Intermediate concentration
Volume haloperidol stock =
(1000 μL * 550 μM)
10 000 μM
10 mM = 10 000 μM
25 μM
22
550 μM
=55 μL stock
1 μCi/1 μl
Radioligand dilution
Need 600 μL tube 1
Final Concentration
Intermediate concentration
110 nmol
1000 ml
85.5 uCi
nmol
x
5 nM
110 nM
1ul
1uCi
x
0.6 ml
x
=
5.6 ul
5.6μL x 1.3 = 7.3μL
*Correction factor (1.3x)
Tube #
1
2
3
4
5
6
7
8
Final Concentration (nM)
5
2
1
0.5
0.2
0.1
0.05
0.02
Volume stock
7.3 μl
200 μl tube 1
100 μL tube 1
50 μL tube 1
50 μL tube 2
50 μL tube 3
50 μL tube 4
50 μL tube 5
Volume
assay buffer
593 μL
300 μL
400 μL
450 μL
450 μL
450 μL
450 μL
450 μL
*Correction factor is used to account for ligand sticking to
plastic; actual ligand concentration is later determined by
spotting a set volume of working material from each tube
onto a Filtermat, measuring dpm, and converting to moles
using specific activity.
Plate map
Radioligand
Full Scale Saturation (550ul)
Concentration
(nM)
Total
1
Non-Specific
2
3
4
5
6
5
1
1
1
1
1
1
2
2
2
2
2
2
2
3
3
3
3
3
3
3
4
4
4
4
4
4
4
5
5
5
5
5
5
5
6
6
6
6
6
6
6
7
7
7
7
7
7
7
8
8
8
8
8
8
8
1) In DeepWell plate:
a. Add 25 μL buffer or Haloperidol
b. Add 25 μL radioligand
c. Add cell membranes (500 uL)
2) Incubate 1 hour
3) During incubation, pre-soak a
UniFilter plate in 0.01% PEI
4) Wash assay plate over cell harvester,
onto UniFilter plate
5) Dry UniFilter plate in 27°C oven
6) Add BackSeal to bottom of UniFilter plate
7) Add scintillation cocktail and TopSeal
8) Measure