Glutamate (NMDA, Non-Selective) Rat Ion Channel [3H] Ifenprodil Binding LeadHunter Assay - TW

Procedure Summary:

Cerebral cortices of male Wistar derived rats weighing 175 ± 25 g are used to prepare glutamate NMDA polyamine receptors in Tris-HCl buffer pH 7.4. A 5 mg‡ aliquot is incubated with 2 nM [³H]Ifenprodil (plus 5 μM GBR-12909 to block non-polyamine sensitive sites) for 120 minutes at 4°C. Non-specific binding is estimated in the presence of 10 μM ifenprodil. Membranes are filtered and washed, the filters are then counted to determine [³H]Ifenprodil 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:

[³H] Ifenprodil

Ligand Kd (nM):

26

Ligand Concentration:

2 nM

Non Specific:

10 µM Ifenprodil

Incubation:

120 min at 4°C

Control Inhibitor:

Ifenprodil

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