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Posted by / 17-Jul-2017 13:46

Yield was 50–75 mg of Ca M per 500 ml of bacterial culture, and Ca M was homogeneous as judged by SDS-polyacrylamide gel electrophoresis.

For purification, cell pellets were resuspended in 50 m EGTA, and protein was detected by the Bradford method.

Ca M was detected by the Bradford method (Bio-Rad), and the appropriate fractions were pooled.

In the second method of Ca M purification, cell pellets that had been resuspended in lysis buffer (50 m MOPS, p H 7.0 (ultra-free spin column, molecular weight 5000 cutoff, Millipore).

All mutagenesis was performed with the Transformer site-directed mutagenesis kit (CLONTECH), and all mutations were verified by DNA sequencing.

Ca M was expressed in the bacterial strain BL21(DE3) (Novagen).

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