Heme-staining of SDS-PAGE gel Theo Laftsoglou Materials: Use rainbow protein markers. Recommended SDS-PAGE Loading buffer For heme staining, prepare your samples in the following loading buffer, vortex and boil them at 90 °C for 20 min or samples may also be treated at 37 °C for 2 h. 6 M Urea 5% w/v SDS 0.1% w/v glycerol 0.05% w/v bromophenol blue Sodium Acetate 0.25M pH5 Methanol TMBD: tetramethylbenzidine dihydrochloride hydrate (stored at 4 °C). Hydrogen peroxide ≥30% (stored at 4 °C). Staining procedure It needs to be done in dark! Cover everything in aluminium foil. 1. Soak the gel in 80 mL 0.25 M Sodium Acetate pH 5 for at least 20 min on the orbital shaker. 2. Prepare in a covered falcon tube: 40 mL methanol & 40 mg TMBD – invert a few times to dissolve. 3. Pour it onto the gel (do not remove sodium acetate buffer) and leave it on the orbital shaker for no more than 15 min. 4. Pour in 400 µL of hydrogen peroxide & cover. Leave the gel on the orbital shaker for at least 60 min (usually overnight). 5. Discard liquid in solvent waste (non-halogenated).