Western Blot Protocol
1. Perform SDS-PAGE on samples to be analyzed, loading 20-30 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the transfer apparatus.
3. Stain the blot using ponceau S for 1-2 minutes to access the transfer of protein. Rinse the blot in water to remove excess stain and mark the lane and molecular weight markers location using a pencil.
4. Rinse the blot in TBST for approximately 5 minutes.
5. Block the membrane using 5% non-fat dry milk in TBST for 1 hour.
6. Dilute the rabbit anti-Beclin primary antibody (NB500-249) in 1% milk-TBST and incubate for 2 hours at room temperature or overnight.
7. Wash the membrane 3x for 10 min each in TBST and apply the diluted rabbit-IgG HRP-conjugated secondary antibody in 1% milk-TBST (as per manufacturer's instructions) and incubate for 1 hour at room temperature.
8. Wash the blot 3x for 10 min each in in TBST.
9. Apply the detection reagent of choice in accordance with the manufacturer's instructions.