Home » Protocols » Calcium binding…
Protocol
Calcium binding assay
Keywords: Calcium binding, SDS-polyacrylamide, gel electrophoresis, protein, calcium binder
A simple method of detecting calcium binding proteins in a protein mixture.
The procedure described can be completed within 24 h and can detect as little as 2 µg of calcium binding protein in the starting sample.
Instructions
-
Calcium binding was performed as described [Maruyama K et. al.] using purified GST-LPCAT1, GST and GST-Secretagogin [Wagner L et. al.] as a positive control.
In brief, 5 µg of each protein were loaded onto a 12% SDS gel and transferred to nitrocellulose. The membrane was washed with binding buffer (60 mM KCl, 5 mM MgCl2 and 10 mM imidazole-HCl, pH 6.8) and incubated 10 minutes in binding buffer with added 1 µCi/ml 45CaCl2.
Non-specifically bound 45Ca was washed with 50% ethanol, and radioactivity was measured on a Phosphorimager.
References
-
Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis ,
Author: Maruyama K, Mikawa T, Ebashi S | Date: 1984 -
Broken reference