Bcl 2 rabbit monoclonal antibody

MCF7 cells were treated with HMWp at IC₅₀ value for 24 h, 48 h and 72 h. Total protein was extracted from the treated cells by using RIPA lysis buffer as described by Yap et al. (2018) (link). Thirty micrograms of protein were separated on 12.5% SDS-PAGE and transferred onto polyvinylidene difluoride membrane. The membrane was blocked with 5% w/v skim milk in TBST (1X TBS, 0.1% Tween-20) for 1 h, and incubated at 4 °C with the corresponding primary antibodies: a β-actin rabbit monoclonal antibody (1:1000), a Bax rabbit monoclonal antibody (1:1000), a Bcl-2 rabbit monoclonal antibody (1:200) and a BID polyclonal antibody (1:1000) (Cell Signaling Technology, Inc., MA, USA), prepared in 5% w/v BSA in TBST. After incubation, membranes were washed with TBST and followed by incubation with HRP-anti rabbit IgG (diluted 1:2000 in 5% skim milk, TBST solution) for 1 h at room temperature. HRP enzyme activity was detected using enhanced luminol-based chemiluminescent substrate (Pierce™ ECL Western Blotting Substrate, Thermo Scientific, USA) and chemiluminescent signal was captured using a CCD-based imaging system (BioSpectrum^® Imaging System, UVP, USA). Quantitative analysis of the signal intensity was performed using ImageJ software (Schneider, Rasb & Eliceiri, 2012 (link)) and the protein expression level was determined by normalization to the internal control β-actin signal intensity.

The cell lysates (10 μl/lane) were separated on a polyacrylamide gel membrane.
After the nonspecific binding sites were blocked with 3 % skim milk, the membrane was treated overnight with Bax Rabbit monoclonal antibody, Bcl‐2 Rabbit monoclonal antibody and LC3A/B Rabbit monoclonal antibody (diluted 1:1000; Cell Signaling Japan). The immunoreactive bands were demonstrated by incubation with anti‐Rabbit IgG‐HRP (IBL) at room temperature for 1 h. Peroxidase activity was visualized with the enhanced chemiluminescence detection system (Amersham). Integrated optical intensities of the immunoreactive protein bands were quantified by imaging and the analysis software Multi Gauge; they were normalized to GAPDH values.