2.5.5 Western blotting
Western blotting was done to separate and identify the size of proteins that react with a specific antibody. After the proteins was transferred onto the PVDF membrane, it was incubated in 5% blocking solution for 1 h at room temperature with constant swirling with belly dancer (Storall Life Science). The blocking of the membrane is to prevent the nonspecific binding of the detection antibodies during next step. After that, washing of membrane was done by using 0.05% TBST for three to five times, each for 10 min. The membranes were then incubated with a primary antibodies (specific polyclonal antibody) as listed in Table 1 with constant shaking, at 4 °C overnight. After incubation, the membranes were washed again with 0.05% TBST three to five times (10 min for each membrane). Incubation of membranes with secondary antibody was performed with peroxidase-conjugated goat anti-rabbit IgG at 1:2000 dilution for 1 h in room temperature. Each of the membranes were again washed with 0.05% TBST for 10 min (three to five times), and 2 mL of ECL™ Prime Western Blotting Detection Reagent (Biorad) was added to detect the peroxidase present.
Types of antibodies Preparation ratio
Β-actin 1:2000
Bax 1:1000 p53 1:1000
Cytochrome c 1:500
Caspase 8 1:2000
Table 1. The list of antibodies …show more content…
The ECL was added to the membranes at 4 mL for each membrane by mixing 1 mL detection solution 1 with 1 mL detection solution 2. The mixture of the reagent was then transferred directly to the side of the membrane that having the protein. The incubation took at 5 min with constant shaking at room temperature. The excess reagent was removed from the container containing membrane and the blot was then placed in an image analyser. Finally, the bands of targeted protein were detected using ChemiUltra Sensitivity filter after 120 sec of the time
Western blotting was done to separate and identify the size of proteins that react with a specific antibody. After the proteins was transferred onto the PVDF membrane, it was incubated in 5% blocking solution for 1 h at room temperature with constant swirling with belly dancer (Storall Life Science). The blocking of the membrane is to prevent the nonspecific binding of the detection antibodies during next step. After that, washing of membrane was done by using 0.05% TBST for three to five times, each for 10 min. The membranes were then incubated with a primary antibodies (specific polyclonal antibody) as listed in Table 1 with constant shaking, at 4 °C overnight. After incubation, the membranes were washed again with 0.05% TBST three to five times (10 min for each membrane). Incubation of membranes with secondary antibody was performed with peroxidase-conjugated goat anti-rabbit IgG at 1:2000 dilution for 1 h in room temperature. Each of the membranes were again washed with 0.05% TBST for 10 min (three to five times), and 2 mL of ECL™ Prime Western Blotting Detection Reagent (Biorad) was added to detect the peroxidase present.
Types of antibodies Preparation ratio
Β-actin 1:2000
Bax 1:1000 p53 1:1000
Cytochrome c 1:500
Caspase 8 1:2000
Table 1. The list of antibodies …show more content…
The ECL was added to the membranes at 4 mL for each membrane by mixing 1 mL detection solution 1 with 1 mL detection solution 2. The mixture of the reagent was then transferred directly to the side of the membrane that having the protein. The incubation took at 5 min with constant shaking at room temperature. The excess reagent was removed from the container containing membrane and the blot was then placed in an image analyser. Finally, the bands of targeted protein were detected using ChemiUltra Sensitivity filter after 120 sec of the time