Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 20)

8.2 Solutions for Zeta-Probe Membrane
Two methods of blocking are given. Method A uses nonfat dry milk as the blocking agent.
Method B uses gelatin and MPO** as the blocking agents. The solutions for the two methods are
not interchangeable. If Method A is chosen, all solutions must be prepared according to Method
A; if Method B is chosen, all solutions must be prepared according to Method B.
TBS, Tris buffered saline, 2 L
Same as nitrocellulose membrane solution.
TTBS, Tween 20, Tris buffered saline, 2 L
Method A. Add 2 ml Tween 20 to 1 L of TBS. This solution is used when nonfat dry milk is the
blocking agent.
OR:
Method B. Add 2 ml Tween 20 and 50 ml MPO to 1 L of TBS. This solution is used when gelatin
and MPO are the blocking agents.
Blocking solution, 100 ml
Method A. Add 5 g of nonfat dry milk to 100 ml of TBS.
OR:
Method B. Add 3 g of gelatin to 100 ml of TBS. Warm to 37°C to dissolve the gelatin; cool before
use.
Antibody buffer, 200 ml
Method A. Add 10 g of nonfat dry milk to 200 ml TTBS. 100 ml is used for the primary antibody
solution and 100 ml is used for conjugate dilution.
OR:
Method B. Add 2 g gelatin to 200 ml TTBS, which already contains 5% MPO. Warm to 37°C to
dissolve gelatin and cool before adding antibody. 100 ml is used for the primary antibody solution
and 100 ml is used for conjugate dilution.
Primary antibody solution, 100 ml
Same as nitrocellulose membrane solution.
Secondary antibody solution, 100 ml
Same as nitrocellulose membrane solution.
Color development solution
Same as nitrocellulose membrane solution.
16