Protocol specific for HIF-1 alpha Antibody (NB100-479)

Western Blot Protocol I

1. Perform SDS-PAGE (3-8% Tris-acetate) on samples to be analyzed, loading 50ug of total protein per lane.
2. Transfer proteins to Nitrocellulose 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 proteins onto the nitrocellulose membrane. Rinse the blot in water to remove excess stain and mark the lane locations and locations of molecular weight markers using a pencil.
4. Rinse the blot in TBS for approximately 5 minutes.
5. Block the membrane using 5% non-fat dry milk in TBS for 1.5 hours.
6. Dilute the mouse anti-HIF-1 alpha primary antibody (NB 100-479) 1:500 in blocking buffer and incubate overnight at 4C.
7. Wash the membrane in water for 5 minutes and apply the diluted rabbit-IgG HRP-conjugated secondary antibody in blocking buffer (as per manufacturer