Concentration: 1 mg/mL or greater
Volume: 1 - 5 µL applied as pendant drop
Buffer: Phosphate buffered saline (PBS): 0.02M phosphate, 0.15M NaCl, pH 7
Drying: Air or oven dry, 5 - 40 °C

Blocking/Capping Agents (in order of blocking efficiency):*

• 0.5% Casein in PBS
• 1% Nonfat dry milk in PBS

Blocking solutions may also contain 0.05% Tween 20 or other non-ionic surfactant which may enhance blocking and will also aid in re-hydrating the membrane if the spotted membrane is stored in a dry state.

*The best method for blocking membranes is 0.5% Hammersten grade casein (BDH 44020 or equivalent) diluted in buffer. Add casein to the buffer and heat on a stir plate to 80 °C or until dissolved; do not boil the solution. Cool to 25 - 40 °C before use. Soak membranes in this solution at room temperature for 30 minutes. Non-fat dry milk can be substituted for the casein, but concentrations may have to be adjusted.

PBS may be sufficient for washing steps. Non-ionic surfactants such as 0.05% Tween 20 may be added to decrease background signal.

Do not use buffers with primary or tertiary amines such as Tris, EDTA or Glycine, as they will compete with amines on proteins for binding to active sites. Do not pre-wet the membrane before immobilization.