A reducing and denaturing step maximizes the immunoprecipitations of m-calpain and I-2(PP2A)/SET: an approach toward antibodies that do not work well in immunoprecipitation.
Immunoprecipitation is an elegant method to isolate a specific protein of interest from a complex protein mixture such as cell lysate. We tried to increase the efficiency of m-calpain immunoprecipitation with anti-m-calpain antibodies directed toward denatured antigens that only work for immunoblotting and immunohistochemistry. We found that a reducing and denaturing step prior to immunoprecipitation greatly potentiates the efficiency of the immunoreaction. This improved method is also applicable for the immunoprecipitation of oncoprotein I-2(PP2A)/SET with antibodies directed toward a synthetic peptide that only work for immunoblotting. Thus, our improved method provides a way to maximize immunoprecipitation when using antibodies that do not work well under conventional immunoprecipitation conditions. Furthermore, the improved method is also suitable for decreasing the contaminating proteins during immunoprecipitation.
Chen
Wei
W
Department of Molecular Biology and Biochemistry, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
Nishikimi
Akihiko
A
Kamata
Tohru
T
Adachi
Yoshifumi
Y