Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 11)

5. Wash each sample well with at least 200 µl of TBS. Pull the wash liquid through by applying
gentle vacuum (flow valve setting 3, Figure 3).
6. After the wells have completely drained, remove the membrane from the apparatus. The best
method for removing the membrane from the Bio-Dot SF apparatus is to leave the vacuum
on following the wash step. With the vacuum applied, loosen the screws and remove the
sample template. Next, turn off the vacuum and remove the membrane.
7. Place the membrane in the blocking solution. The blocking solution used is dependent on the
type of membrane used. Nitrocellulose is blocked in gelatin, BSA, or non fat dry milk. Zeta-
Probe membrane is blocked in either nonfat dry milk or gelatin and 1-methyl-2-pryrrolidinone
(MPO) (see Section 10, solution preparation). All solution volumes should be at least 0.5
ml/cm
2 of membrane. Larger volumes may be used for convenience. Gently agitate the
solution using a shaker platform. Continue the incubation for 30 minutes to 1 hour.
8. Remove the membrane from the blocking solution and transfer to a dish or tray containing
Tween, Tris-buffered saline (TTBS). Wash for 5 minutes with gentle agitation. Decant the wash
solution. Repeat the wash step.
9. Transfer the membrane from the TTBS wash solution to a dish or tray containing the primary
antibody solution. Incubate 1 to 2 hours with gentle agitation. Overnight incubation may be
desirable, since longer incubation periods may increase the sensitivity of detection in some
situations.
10. Remove unbound primary antibody by washing the membrane in TTBS for 5 minutes with
gentle agitation. Decant the wash solution and repeat the wash step.
11. Transfer the membrane to the secondary antibody conjugate solution.
12. Transfer the membrane from the conjugate solution and wash it in TTBS for 5 minutes.
Decant the buffer and repeat the wash step. Prior to color development, do a final 5 minute
wash in TBS to remove residual Tween 20 detergent from the membrane surface.
13. Do the color development reaction according to the Immun-Blot assay kit instruction manual.
If performing autoradiography, remove the membrane, dry it on a filter paper, wrap it with
plastic wrap, and expose it to X-ray film.
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