BB3 Human Bombesin GPCR Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [¹²⁵I] (D-Tyr⁶,beta-Ala¹¹,Phe¹³,Nle¹⁴)-Bombesin (6-14) to human bombesin BB3 receptors. Balb 3T3 cells stably transfected with a plasmid encoding human bombesin BB3 receptors are used to prepare membranes in modified HEPES-KOH pH 7.4 buffer using standard techniques. A 2.5 μg‡ aliquot of membrane is incubated with 0.1 nM [¹²⁵I] (D-Tyr⁶,beta-Ala¹¹,Phe¹³,Nle¹⁴)-Bombesin (6-14) for 60 minutes at 25°C. Non-specific binding is estimated in the presence of 1 μM [D-Phe6, b-Ala11, Phe13, Nle14]-Bombesin (6-14). Membranes are filtered and washed 3 times and the filters are counted to determine [¹²⁵I] (D-Tyr⁶,beta-Ala¹¹,Phe¹³,Nle¹⁴) Bombesin (6-14) specifically bound. Compounds are screened at 10 μM.
Note: ‡Membrane protein may change from lot to lot, the concentration used will be adjusted if necessary.

Ligand:

[¹²⁵I] (D-Tyr⁶,beta-Ala¹¹,Phe¹³,Nle¹⁴)-BN (6-14)

Ligand Kd (nM):

0.21

Ligand Concentration:

0.1 nM

Non Specific:

1 µM (D-Phe⁶,b-Ala¹¹,Phe¹³,Nle¹⁴)-BN (6-14)

Incubation:

60 min at 25°C

Control Inhibitor:

(D-Phe⁶,beta-Ala¹¹,Phe¹³,Nle¹⁴)-Bombesin (6-14)

Criteria For Significance:

≥ 50% of max stimulation or inhibition

Test Concentration / Dose:

Suggested concentration for initial testing is 10 uM

Test Sample Requirements:

Minimum for 1) Screen: 25 μl of 10 mM stock -OR- 1 mg (pre-weighed). 2) Dose Response: 60 μl of 10 mM stock -OR- 1 mg (pre-weighed).

Minimum Order Quantity:

2