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6. Purification of CaM from bovine tissues using melittin-Sepharose/organomercurial agarose chromatography. Tissue supernatants from bovine brain, heart, liver, and testis were prepared as described under method 1. After adsorption and elution of melittinSepharose, a portion of the eluate was removed prior to application to organomercurial agarose. Samples of melittin-Sepharose eluate (20/~1) and organomercurial agarose passthrough (40 p,1), respectively, were precipitated with trichloroacetic acid prior to SDS-gel electrophoresis.

Purification of Calelectrins from Bovine Tissues Reagents Acetone powders derived from bovine liver, brain, pancreas, heart, testes, lung (Sigma Chemical Company, St. 4 Acetone powder extraction buffer: 0. 4 Either fresh tissues or acetone powders commercially available from Sigma Chemical Company can be used as starting materials. The following tissues have been used with very good or satisfactory yields: liver, kidney, testis, pancreas, adrenal gland, heart, brain, and smooth muscle. Because of its size and content the highest quantities of calelectrins are obtained from liver.

R. Scully, V. L. Smith, E. Bernicker, and J. R. Dedman, this volume [2]. METHODS IN ENZYMOLOGY, VOL. 139 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved. 30 I S O L A T I O N / C H A R A C T E R I Z A T I O N OF C a - B I N D I N G PROTEINS [3] samples. In addition, the anti-35K antibody showed cross-reactivity with two more proteins (MW 47K and MW 27K) in the cytosol. These results suggest that the 35K and 67K calcimedins have some common epitopes. Moreover, the immunoreactive 47K and 27K species may also share common epitopes with the 35K calcimedin.

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