PDSP - Adrenergic

   
 
For each receptor class, one binding assay is described in detail. All binding assays are carried out with an incubation time of 90 MIN/RT (RT=room temp). All other binding assays are performed in a similar manner, although the buffers used differ from assay to assay. The table below gives the (1) radioligand; (2) assay buffer; (3) unlabelled reference ligand; and (4) references to demontrate expertise.

Adrenergic Radioligand Assay buffer Unlabelled ligand as reference compound References
Alpha1A 3H-Prazosin (1nM) A uripadil Roth lab standard protocol
Alpha1B 3H-Prazosin A corynanthine Roth lab standard protocol
Alpha2A 3H-Cholidine (2nM) A oxymetazoline Roth lab standard protocol
Alpha2B 3H-Cholidine (2nM) A prazosin Roth lab standard protocol
Alpha2C 3H-Cholidine (2nM) A prazosin Roth lab standard protocol
Beta1 125I-Pindolol A Atenolol Roth lab standard protocol
Beta2 125I-Pindolol A Atenolol Roth lab standard protocol
Beta3 125I-Pindolol A Atenolol Roth lab standard protocol

Assay buffers:

A=50 mM Tris-Cl, 0.5 mM EDTA, 5 mM MgCl2, pH=7.4
B=50 mM Tris-Cl, 154 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, pH=7.4
C=50 mM Tris-Cl, 0.5 mM EDTA, 5 mM MgCl2, 0.01% ascorbic acid, 10 uM pargylline; pH=7.4
D=50 mM Tris-Cl, pH=7.4

Assay description for Adrenergic receptors:

Set up 96-well plate as follows:

UNK #1 UNK #1 UNK #2 UNK #2 UNK #3 UNK #3 UNK #4 UNK #4
UNK #5 UNK #5 UNK #6 UNK #6 UNK #7 UNK #7 UNK #8 UNK #8
UNK #9 UNK #9 UNK #10 UNK #10 UNK #11 UNK #11 UNK #12 UNK #12
               
               
               
               
               
               
STANDARD #1 STANDARD #1 STANDARD #2 STANDARD #2 TOTAL BINDING TOTAL BINDING TOTAL BINDING TOTAL BINDING
STANDARD #1 AT 1 nM STANDARD #1 AT 1 nM STANDARD #1 AT 3 nM STANDARD #1 AT 3 nM STANDARD #1 AT 10 nM STANDARD #1 AT 10 nM STANDARD #1 AT 30 nM STANDARD #1 AT 30 nM
STANDARD #1 AT 100 nM STANDARD #1 AT 100 nM STANDARD #1 AT 300 nM STANDARD #1 AT 300 nM STANDARD #1 AT 1000 nM STANDARD #1 AT 1000 nM TOTAL COUNTS ADDED / PLATE TOTAL COUNTS ADDED / PLATE

1 For 3H-ligands use 1-2 nM final concentration of radioligand; for 125I-radioligands, use 0.05-0.1 nM final concentration.
2 Pipette in following order:
--Binding buffer
--Radioligand
--Cold unknown ligand
--Cold reference ligand
--Membranes
3 Incubate at RT as listed in binding assay table above.
4 At the end of the incubation, harvest onto pre-soaked (0.3% polyethyleneimine) GF/C filters using 96-well harvester. Wash with 3 quick washes with ice-cold harvest buffer.
5 Remove filters and air dry overnight.
6 Count in 96-well counter using 100 ul of scintillant/well.
7 Calculate Ki value of reference compound using LIGAND Program.
8 Calculate % inhibition using following formula:
% Inhibition=Counts bound at 10 uM concentration of unknown compound x 100
                                Total specific counts