CRHR1(CRFR1) Binding Assay
:
(1) Receptor Incubation Buffer(RIB) (PH=7.4)
50mM Tris ( 7.88g Tris HCl/l)
2mM EGTA (760 mg EGTA/l)
10mM MgCl2 (2.03g MgCl2/l)
Add 0.1% BSA and 1-10% Proteinase Inhibitor Cocktail before use
(2) CP-154,526-1(Antalarmin)
Stocking Solution: 1.5mg/1.5ml(50%EtOH/5mM HCl)=1mg/1ml=2745uM(MW=364.35)
Take 1ul of stocking solution+9ul of RIB working solution=274.5uM( 1:10 diluted)
Take 3.64ul of diluted to 1ml of RIB working solution ( final concentration is 1 uM)
(3) Sauvagine(American Peptide Company, Catalog#34-7-11)
Stocking Solution: 500uM(500X)
Take 1.4ul of 500uM Sauvagine to 700ul of RIB working solution(final concentration is
1 uM)
(4) 125I-Sauvagine(Du Pont New England Nuclear, Catalog#NEX306010UC)
Each reaction needs 200PM 125I
Specific activity of 125I: 2200Ci/mmol
y=uCi/tube(reaction)=0.044uCi/tube
125I concentration=10uCi/200ul=0.05uCi/ul
Each tube needs 0.044/0.05=0.88ul
If there are 20 reactions, take 20x0.88=17.60ul 125I to 2000ul RIB working solution
(5) PBS 0.01%TritonX-100
20ml of 1X PBS+2ul of TritonX-100
(1) Total Binding(CRHR1+CRHR2)
100ul membrane protein+100ul RIB working solution+ 100ul 125I
(2) CRHR2 Binding
100ul membrane protein+100ul Antalarmin in RIB working solution+ 100ul 125I
(3) Non-Specific Binding
100ul membrane protein+100ul Sauvagine in RIB working solution+ 100ul 125I
CRHR1 Binding=Total Binding - CRHR2 Binding - NonSpecific Binding
300ul reaction mixture performed in 1.5ml eppendorf tube

100ul RIB working solution
or
100ul membrane(50ug)+ 100ul Antalarmin in RIB working solution + 100ul 125I
or
100ul Sauvagine in RIB working solution

mix well and spin down

incubate 2hr at room temperature
centrifuge 10min at 12000g

aspirate supernatant and wash pellet with ice cold PBS 0.01% TritonX-100

centrifuge 10 min at 12000g

aspirate supernatant

cut the tube just above the resulting pellets, place into polystyrene tubes

monitor for radioactivity in gamma counter