User Manual

8.2 Solutions for Zeta-Probe Membrane for Protein Applications

When immobilizing antigen onto the Zeta-Probe

membrane, the immunoassay must be

performed in a separate container following removal of the membrane from the Bio-Dot

apparatus. Two methods of blocking are given: Method A uses nonfat dry milk (Jerome

and Jiehning 1986, Johnson et al. 1984) as the blocking agent. Method B uses gelatin and

1-methyl-2-pyrrolidinone (MPO) as the blocking agents. The solutions for the two methods

are not interchangeable. If Method A is chosen, all solutions must be preparied 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 3 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

with stirring. 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 that 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 antibody buffer solution.

Secondary antibody solution, 100 ml

Same as nitrocellulose membrane antibody buffer solution.

Color development solution

The specific chemicals and buffers are dependent on the enzyme conjugate being

used. See the Bio-Rad Immun-Blot

assay kit instruction manual for details on how to

make the appropriate solution.