GABAA Receptor Binding Assay Protocol

Adapted by Peterson S.

Buffers:

Homogenization Buffer: 0.32 M sucrose, pH 7.4; Keep at 4° C
Binding Buffer: 50 nM Tris-HCl; pH 7.4; Keep at 4° C

Membrane Preparation:

  1. Homogenize rat brains in 20 ml/g of homogenization buffer to grams of tissue.
  2. Centrifuge at 1000 x g for 10 min at 4° C
  3. Centrifuge supernatant at 140,000 x g for 30 min at 4° C
  4. Disperse pellet in 4° C deionized water
  5. Homogenize on high speed setting for two 10 s bursts, 10 s apart
  6. Centrifuge at 140,000 x g for 30 min at 4° C
  7. Resuspend pellet in binding buffer at 4° C
  8. Centrifuge at 140,000 x g for 30 min at 4° C
  9. Repeat steps 7 and 8 twice.
  10. Resuspend pellet in binding buffer at 4° C
  11. Store at -70° C until day of binding assay

Binding Assay:

  1. Thaw tissue, wash with binding buffer twice by centrifugation at 140,000 x g for 30 min at 4° C
  2. Resuspend pellet in binding buffer
  3. Add pellet to binding assay, 0.1-0.2 mg of protein to each well; 10-20 mg per plate.
  4. Use 10 mM GABA for non-specific binding determination
  5. Assay with 5 nM concentration of [3H] muscimol at 4° C for 45 min
  6. Terminate with 4° C standard wash buffer (50 mM Tris-HCl, pH 7.4)
  7. Quantify radioactivity by liquid scintillation spectrometry


  

Protocol adapted from Chronic ethanol administration alters the modulatory effect of 5alpha-pregnan-3alpha-ol-20-one on the binding characteristics of various radioligands of GABAA receptors.
A.K. Mehta, M.K. Ticku   Brain Res. 1998 Sep 14;805(1-2):88-94