Western Blot

Following Procedure was followed for Western Blot:

•PVDF membrane was cut equal to the size of the gel and charged by soaking into methanol.

•Prechilled transfer buffer was poured in a glass tray and cassette was put in it with black side down

•transfer assembly was arranged from bottom to top as follows: foam pad, 2-3 whatmann sheets, gel, membrane, 2-3 whatmann sheets, foam pad. Air bubbles were avoided.

•Tranfer was carried out at 150mA for 1hr in prechilled 1X transfer buffer.

•Membrane was kept in 5% blocking solution for 1-2 hrs.

•Incubated overnight in 1º Ab.

•Washed with TBST thrice (15 min each)

•Incubated in 2º Ab for 1hr

•Washed with TBST thrice (15 min each)

•Developed using ECL/ECL prime (GE Healthcare)