Simple immunoassays for semiquantitative determination of calmodulin (CaM) and anti-CaM. Applications to affinity column chromatography of anti-CaM antibodies and estimation of CaM in synaptosomal membranes from rat cerebral cortex.

ABSTRACT

Dot-immunobinding assay for anti-calmodulin (CaM), and immunoblot assay for CaM have become feasible by proper choice of a fixative, i.e., formaldehyde vapor. The protocol for these simple and convenient immunological procedures is presented. Dot-immunobinding assay could be accomplished in 3 h, and immunoblot assay in 8 h. Activities of anti-CaM antibodies in fractions eluted from CaM affinity column were monitored by immunobinding assay. Semiquantitative immunoblot analysis was used to estimate CaM present in various fractions during preparation of EGTA-washed lysed synaptosomal membranes from rat cerebral cortex. CaM content in the final preparation was estimated at lower than 3 micrograms/mg of membrane protein.