| Assay Method Information | |
| | Creating an a7 Nicotinic Acetylcholine Recognition Domain from the Acetylcholine-binding Protein |
| Description: | Assay Description 2 Assays used to generate Ki or EC50 values. 1) SPA Assay - Quick screen binding assays were performed using 100 ul of 0.2 mg/ml anti-mouse SPA antibody-binding beads (Amersham Biosciences) mixed with 33.6 u of monoclonal anti-FLAG M2 antibody from mouse (Sigma) in 0.1 M NaPO4 buffer, pH 7.0 (SPA mixture). Media (5 ul) collected 2 days after transfection were added to the mixture and finally 10 ul of ()-[3H]epibatidine (PerkinElmer Life Sci- ences) was added at a final concentration of 10 nM. Solutions were each counted for 1 min on either a Beckman LS 6500 liquid scintillation counter or Wallac 1450 Microbeta Trilux. Negative and positive controls consisted of media (5 ul) from the respective transfections. Nonexpressing mutants were transfected and verified de novo in triplicate. Western immu- noblots were also performed upon media (15 ul) from transfec- tions to verify the lack of expression. Competition curves and direct binding were measured fol- lowing the pro |
| Affinity data for this assay | |
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