Bcl 2 elisa kit

Bcl-2 and Bax protein expressions were analyzed in all samples by enzyme-linked immunosorbent assay (ELISA). This was undertaken using monoclonal antibodies against Bax (Ab-1) (DRG^® Human Bax ELISA Kit) and anti-Bcl-2 (Zymed^® Bcl-2 ELISA Kit) according to the manufacturer’s instructions.

The level of the anti-apoptotic marker and apoptotic marker BAX were assessed using Bcl-2 Elisa kit and human Bax ELISA Kit purchased from Zymed laboratories, invitrogen and Cloud-Clone Crop. (Katy, TX, USA), respectively, following the manufacturer’s instructions. Briefly, Treated MCF-7 cell lysate with 250 nM of compound 12c were prepared, and equal amount of cell lysates were loaded and propped with specific antibodies. The samples were measured and analysed at 450 nm in ROBONEK P2000 ELISA reader⁵² (link). In Vitro Caspase-9 Activation Assay was performed using human active caspase-9 Invitrogen EIA kit according to the manufacturer’s instructions. Compound 12c at concentrations of 50 and 250 nM and CA-4 (50 nM) were prepared in dH₂O up to a final volume of 50 µL/well followed by addition of 5 µL of active caspase-9. Following, the cells were mixed and 50 µL of the Master Mix was added to each well and allowed to react at 37 °C for 1 h. The fluorescence intensity of the test samples was recorded and analysed in a fluorescence plate reader at 400 nm excitation and 505 nm emissions. All experiments were conducted in triplicates.

The quantitative determination of BAX and BCL-2 proteins was carried out after 24 h treatment by using Human BAX ELISA kit (DRG Instruments GmbH, Marburg, Germany) and Zymed^® Bcl-2 ELISA Kit, respectively, according to the manufacturers’ instructions (Fahmy and Aldawsari, 2020 (link); Md et al., 2020 (link)). Untreated cells were considered as a control. MEL, FLV-R, or FLV-MEL nano-conjugates were used at a sub-toxic concentration (IC10).