[35S]GTPgS Binding Assay by Beads Counter:
 

Receptor:

rKOR cell line membrane fragments, detach cell (PBS), centrifuge (4000 rpm/15 min), then washing again with Standard binding buffer, pellets can be saved at -80°C. For each GTPgS assay plate, 4-5 pellets needed (1pellet/dish).

Drugs:

Agonist:: pdsp3251   rb-64   rb-48   sala  
Agonist mw 464   489.5   466.4   432.5  
solubility DMSO/H2O   DMSO/H2O   DMSO/H2O   DMSO/H2O  
Agonist assay 25 mM 25 mM 25 mM 25 mM
multiplicity 4 X 4 X 4 X 4 X
plate 1   1   1   1  
Agonist volume 0.5 ml 0.5 ml 0.5 ml 0.5 ml
Agonist stock 1 mg/ml 2 mg/ml 2 mg/ml 2 mg/ml
Agonist stock added 23.2   12.2   11.7   10.8  
buffer added 477   488   488   489  
Antagonist naloxone   naloxone   naloxone   naloxone  
Antagonist MW 364   364   364   364  
Antagonist stock 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml
NS mix1 added 18.2   18.2   18.2   18.2  
NS mix1 buffer 459   470   470   471  
GTPg Assay 10 mM 10 mM 10 mM 10 mM
GTPg Stock 1 ml 1 ml 1 ml 1 ml
NS mix2 added 20   20   20   20  
NS mix2 buffer 457   468   468   469  

Other Materials:

[35S]GTPgS (-80°C cell pellet refrig 3rd tower, 10ml stock, final assay concentration 0.05-0.3nM);
Assay stock multiplicity decay factor days plates volume stock added buffer needed
150 1 4 1.1719 21 1 5520 3.88 5516
pM mM X     plate ml ml ml
*Example for calculation: 150pM [35S]GTPgS: Stock is 1 µM, 4X is 0.6nM. So 1 µM: 0.6nM =1:1667, checking decay factor 0.7576 (35 days behind), 1667*0.7576=1263, so 21.0 ml/1263=16.6 µl.
GDP (-20 next to high speed refrig, sigma Aldrich bottle, need to weight fresh);
Assay stock MW multiplicity plates volume stock added buffer needed
20 1 443.2 2 5520 98 5422
mM mg/ml   X plate ml ml ml
*Example for calculation: 20 µM GDP (4X=80 µM): Volume*10mg/ml/443.2(MW)=80 µM*21 ml, Volume=74.46 µl.
GTPgS (-80 ºC standing next to puncher, 1mM stock);
GTP is weight out and made a stock 1mM, MW 563.
Naloxone (KOR antagonist).

Procedure:

Set up assay in 96 well beads counter plate. 50 µl drug solution, 50 µl [35H]GTPgS, 50 µl membrane+GDP (15min incubation), incubating for 15min, then 50 µl beads (110 µl stock solution/ 5.5 ml buffer). Shake the mixture for half an hour, then incubate 15 min. Gently spin down, 1000 rpm/15min, ready for counting.

  1. Make drug dilution (4X) and add 50 µl to each well, and the last two columns are corresponding antagonist or GTPgS.
  2. Make membrane (receptor) premix in GTPgS buffer, plus freshly made GDP, incubate for 15min.
  3. Add 50 µl [35H]GTPgS in each well of 96-well plates.
  4. Add 50 µl membrane+GDP premix to each well, incube for 15 min.
  5. Add 50 µl beads solution to each well of 96-well plates.
  6. Shake the plate for 30 min and incubate for another 15 min at RT.
  7. Gently centrifuge at 1000 rpm (~270g) 5 min. Counting. At least count twice, usually analyzing the first set of data.

    8. Plate layout:

    0 0.1pM 1pM 10pM 0.1nM 1nM 10nM 100nM 1mM 10mM N.S. N.S.

    GTPgS Buffer:

    50 mM Tris·HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA

    Buffer KOR (GTPs) MW Stock 1 L 0.5 L Final Conc.
      ________________________________________________
    NaCl   2M 50 ml 25 mL 100 mM
    MgCl2   1M 10 ml 5 mL 10mM
    EDTA   0.5M 2 ml 1 mL 1 mM
    Tris-HCl   1 M 50 mL 25 mL 50 mM