Glutamate (Non-Selective) Rat Ion Channel [3H] L-Glutamic Acid Binding LeadHunter Assay - TW

Procedure Summary:

Whole brain (except cerebellum) membranes of male Wistar derived rats weighing 175 ± 25 g are prepared in Tris-HCl buffer pH 7.4. A 1 mg‡ aliquot of membrane is incubated with 3.75 nM [³H]L-Glutamic acid for 30 minutes at 37℃. Non-specific binding is estimated in the presence of 50 μM L-glutamic acid. Membranes are filtered and washed, the filters are then counted to determine [³H]L-Glutamic acid 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] L-Glutamic acid

Ligand Kd (nM):

290

Ligand Concentration:

3.75 nM

Non Specific:

50 µM L-Glutamic acid

Incubation:

30 min at 37°C

Control Inhibitor:

L-glutamic acid

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