Chemiluminescence detection kit for hrp

Cells were lysed with lysis buffer containing 2% SDS, 0.1% bromophenol blue, 10% glycerinum, 1.5% DTT (dithiothreitol), and 0.1 M Tris-HCl (pH 6.8). Cell lysates were quantitated by a BCA protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The boiled lysates were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes and blotted with 5% non-fat milk (dissolved in TBST). Then, the membranes were incubated with the indicated primary antibodies at 4 °C overnight and then with the horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, Shanghai, China). TBST (TBS with 0.1% Tween-20) was used to wash the membranes following antibody incubation. Ultimately, the membranes were visualized in an Amersham Imager 600 system (GE Healthcare Life Science, Shanghai, China) with a Chemiluminescence Detection Kit for HRP (Biological Industries, Cromwell, USA).

Generally, cell lysates were collected with 1× loading buffer containing 2% SDS, 0.1% bromophenol blue, 10% glycerinum, 1.5% DTT (dithiothreitol) and 0.1 M Tris‐HCl (pH 6.8). The cells lysates were boiled and separated by SDS‐PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Bio‐Rad, Shanghai, China), and then blocked with 5% non‐fat milk (dissolved with TBST). The membranes were incubated with the indicated primary antibodies at 4°C overnight and then incubated with the suitable secondary antibody conjugated with horseradish peroxidase (HRP) (111‐035‐003, Jackson Immuno Research, USA) at 37 °C for 2 h. Ultimately, the membranes were visualised in an Amersham Imager 600 system (GE Healthcare Life Science, Shanghai, China) with a Chemiluminescence Detection Kit for HRP (Biological Industries, Cromwell, USA, 20‐500‐120; FDbio, china, FD8030) to determine protein expression.

Cell lysates were collected with loading buffer containing 2% sodium dodecyl sulfate, 10% glycerinum, 0.1% bromophenol blue, 1.5% dithiothreitol, and 0.1 M Tris-HCI (pH 6.8). The cell lysates were boiled loaded on SDS-PAGE gels transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, Shanghai, China), and blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween^® 20 detergent (TBST). The blots were incubated with the indicated primary antibodies at 4 °C overnight. The next day, the blots were washed with TBST and incubated with the suitable secondary antibody (111-035-003, Jackson Immuno Research, West Grove, PA, USA) at room temperature for 2 h. The blots were then visualized on a Bio-Rad ChemiDOC Touch Imaging System (Bio-Rad Laboratories, China) with a Chemiluminescence Detection Kit for HRP (20-500-120, Biological Industries, Cromwell, CT, USA; FD8030, FDbio Science, China) for detection.