User Manual

4.2 Special Protein Blot Applications

1. Soluble enzyme substrate reactions and quantitations

Perform an immunassay as described in Section 4.1. Prior to color development,

disconnect the vacuum and close the flow valve. Add an equal volume of substrate

solution to all wells. Visualize positive reactions and record. For quantitation, withdraw

equal aliquots of the soluble substrate reactant from each well and transfer to a plastic

disposable microplate. Quantitate using Bio-Rad’s Model 620 video densitometer.

2. Assay for particular antigen or target cell antigen

a. Place a prewetted filter paper (Whatman GF/B) in the Bio-Dot apparatus. Attach the

sample template and tighten the screws. Fill all the wells with buffer and apply a

vacuum. With the vacuum on and the buffer draining, retighten the screws. The

presence of buffer while applying vacuum will help prevent the filter paper from

breaking. When the buffer is gone, turn off the vacuum and close the flow valve.

b. Add 50 µl fetal bovine serum (FBS), 10% v/v in blocking buffer. Allow the FBS buffer

to incubate for 10 minutes, then open the flow valve and filter through by gravity.

c. Add approximately 12,500 target cells in 50 µl FBS buffer to each well. Gently pull

the solution through the membrane by attaching tubing to the flow valve to increase

the hydrostatic pressure (see Section 3.2). Perform three washes with TBS buffer

using tubing rather than vacuum to speed the flow rate.

d. Perform antibody incubations as described in Section 4.1 for protein immunoassays.