Irdye 800 cw conjugated anti rabbit igg and irdye 680 cw conjugated anti mouse igg secondary antibodies

Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (pH 7.4) (50 mM Tris-HCl, 0.5% Nonidet P-40, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1% protease inhibitor mixture, 1 mM sodium orthovanadate, and 1 mM sodium fluoride). Proteins were separated on an SDS-PAGE gel and transferred onto nitrocellulose membranes, followed by blocking in 0.1% phosphate-buffered saline (PBS)–Tween (PBST) with 5% bovine serum albumin (BSA) and incubation with primary antibodies at 4°C overnight. Detection was performed with IRDye 800 CW-conjugated anti-rabbit IgG and IRDye 680 CW-conjugated anti-mouse IgG secondary antibodies (Li-Cor) or horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Immunoreactive bands were visualized using an Odyssey infrared (IR) imaging system (Li-Cor). The bands were quantified using Quantity One software (Bio-Rad).

Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (pH 7.4) (50 mM Tris-HCl, 0.5% Nonidet P-40, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1% protease inhibitor mixture, 1 mM sodium orthovanadate, and 1 mM sodium fluoride). Proteins were separated on an SDS-PAGE gel and transferred onto nitrocellulose membranes, followed by blocking in 0.1% phosphate-buffered saline (PBS)–Tween (PBST) with 5% bovine serum albumin (BSA) and incubation with primary antibodies at 4°C overnight. Detection was performed with IRDye 800 CW-conjugated anti-rabbit IgG and IRDye 680 CW-conjugated anti-mouse IgG secondary antibodies (Li-Cor) or horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Immunoreactive bands were visualized using an Odyssey infrared (IR) imaging system (Li-Cor). The bands were quantified using Quantity One software (Bio-Rad).