Horseradish peroxidase hrp coupled mouse igg kappa binding protein m iggκ bp hrp

For the generation of whole-cell lysates from cultured cells growing in wells of 24-well plates, at first the growth medium was removed and then the cell monolayers were frozen and thawed again by resuspension in 2.5-fold pre-heated (95°C) Laemmli buffer. Cell lysates were then heated for 10 min at 95°C, followed by SDS-PAGE and transfer of the lysate proteins by Western blotting onto a nitrocellulose membrane. For the immunodetection of non-glucosylated Rac1 and GAPDH, primary mouse anti-Rac1 (#610651; clone 102; BD Biosciences, Heidelberg, Germany) and mouse anti-GAPDH (sc-365062; G-9; Santa Cruz Biotechnology, Dallas, USA) antibodies were used, respectively. Horseradish peroxidase (HRP)-coupled mouse IgG kappa-binding protein (m-IgGκ BP-HRP; Santa Cruz Biotechnology, Dallas, USA; sc -516,102) and HRP-coupled goat anti-mouse IgG (H+L) secondary antibody (#31430; Thermo Fisher Scientific, Waltham, USA) were used for the generation of antibody signals that were detected by the enhanced chemiluminescence (ECL) reaction.

Whole-cell lysates from cells growing in the wells from a 24-well plate were generated directly in wells by removing the growth medium and resuspending cell monolayers in 2.5-fold pre-heated Laemmli buffer (typically 30 μl per well). Lysate samples were heated at 95°C for 5 min, prior to SDS-PAGE and Western blotting of lysate proteins onto a nitrocellulose membrane. For the immunodetection of non-glucosylated Rac1 and GAPDH, primary mouse anti-Rac1 (clone 102; BD Biosciences, Heidelberg, Germany; #610651) and mouse anti-GAPDH (G-9; Santa Cruz Biotechnology, Dallas, United States; sc-365062) antibodies were used, respectively. Horseradish peroxidase (HRP)-coupled mouse IgG kappa binding protein (m-IgGκ BP-HRP; Santa Cruz Biotechnology, Dallas, United States; sc-516102) was used for developing antibody signals by the enhanced chemiluminescence (ECL) reaction.