Modified and simplified western blotting protocol: use of intermittent microwave irradiation (IMWI) and 5% skim milk to improve binding specificity.
A modified western blotting protocol was developed to increase the binding specificity of antigens and antibodies, using intermittent microwave irradiation (IMWI) with seven antibodies and two cell lines. The method was based on IMWI of the blotting membrane in the immunoblotting step using 5% skim milk as the diluting buffer. For some antibodies against p53, CDK4 and cyclinE, there were no distinct differences between the IMWI(+) and IMWI(-) counterparts; but improvement over the standard protocol was noted in both. For some antibodies, such as the polyclonal antibody against tubulin and the monoclonal antibodies against beta-tubulin, cyclinA and cyclinB1 (which were otherwise difficult to obtain good results with), IMWI was extremely effective, resulting in clear, specifically binding bands and a clean background. Moreover, the times were reduced from 8 to 3 h. Both the IMWI(+) and IMWI(-) protocols can be applied as simple, rapid and highly specific detection techniques for applications with various antigens, reducing background 'noise' to a minimum.
Li
Wei
W
First Department of Pathology, Toyama Medical and Pharmaceutical University, Toyama, Japan.
Murai
Yoshihiro
Y
Okada
Eikichi
E
Matsui
Kazuhiro
K
Hayashi
Shinichi
S
Horie
Masaki
M
Takano
Yasuo
Y
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000528:radiation effects*; D000918:Antibody Specificity / Q000276:immunology / Q000528:radiation effects*; D015153:Blotting, Western / Q000379:methods*; D002021:Buffers; D002417:Cattle; D006801:Humans; D008872:Microwaves; D008892:Milk / Q000737:chemistry; D011506:Proteins / Q000276:immunology / Q000528:radiation effects*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D002021:Buffers; D011506:Proteins