A simple quantitative assay of 125I-labeled α-bungarotoxin binding to soluble and membrane-bound acetylcholine receptor protein
✍ Scribed by Ronald A. Kohanski; John P. Andrews; Pierre Wins; Mohyee E. Eldefrawi; George P. Hess
- Publisher
- Elsevier Science
- Year
- 1977
- Tongue
- English
- Weight
- 535 KB
- Volume
- 80
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
This method involves the irreversible formation of a complex between ?labeled cu-bungarotoxin and the acetylcholine receptor in either its membranebound or purified state. The separation of the labeled toxin-receptor complex from unreacted toxin is accomplished by chromatography on carboxymethylcellulose cation-exchange resin. The method described was developed to satisfy the following experimental requirements that could not be dealt with in their entirety by employing any of the published methods: (i) the complete recovery of reacted and unreacted species in relatively small volumes; (ii) an efficient and precise isolation of the specific and irreversible '*Wabeled cr-bungarotoxin-receptor complex when the complexation reactions demand a large excess of unlabeled a-bungarotoxin for quenching (a 20-fold molar excess of unlabeled over labeled toxin); (iii) this isolation of the toxin-receptor complex allows one to determine the protein concentrations in the samples, a necessity in experiments covering a wide range of receptor concentrations: (iv) a consistent low blank for binding site concentrations ranging over two or three orders of magnitude: (v) simplicity and rapidity.