User Manual

6. Adjust the flow valve so that the vacuum chamber is exposed to air. Add 200–400 µl of
the Tween, tris-buffered saline (TTBS) wash solution to each well. Adjust the flow valve to
the vacuum position and pull the wash solution through the membrane. Disconnect the
vacuum as soon as the wash solution has drained from all the sample wells. Repeat the
wash step. If the membrane is to be removed from the apparatus prior to performing an
immunoassay, remove it at this point. Otherwise, proceed to step 7. Note: For better
results with Zeta-Probe, use 0.3% Tween 20.
7. Open the flow valve to air. Add 100 µl of primary antibody solution to each sample well.
Allow gravity filtration to occur until the antibody solution has completely drained from the
sample wells (approximately 30–40 minutes).
8. Apply vacuum to the apparatus to remove any excess liquid from the sample wells.
9. Open the flow valve to the atmosphere and add 200–400 µl of TTBS wash solution to
each well. Apply vacuum until the wash solution is drained from the wells. Repeat for a
total of three wash cycles.
10. With the vacuum off and the flow valve open to air, add 100 µl of secondary antibody
solution to each well. Allow gravity filtration to occur (30–40 minutes) until all solution has
drained from the wells.
11. Turn the vacuum on and drain the wells. Add 200–400 µl of TTBS wash solution to each
well and drain completely. Repeat for a total of two washes.
Note: At this point, the membrane is ready for development. Color development of
enzyme conjugated antibodies can be performed in the apparatus or in a separate
reservoir. If performing autoradiography, remove the membrane, dry it on a filter paper,
wrap it with plastic wrap, and expose it to X-ray film. The best method to remove the
membrane from the Bio-Dot apparatus is to leave the vacuum on following the last wash
step. While the vacuum is on, loosen the screws and remove the sample template. Turn
off the vacuum and remove the membrane.
12. For color development in a separate vessel, remove the membrane and place it in the
color development vessel. Wash the membrane twice with TBS to remove excess Tween
20. Prepare the color development solution, and incubate the membrane in the solution.
Gently agitate the solution until development is complete. When the reaction has
developed, remove the membrane and rinse it in distilled water to stop the reaction. Place
the membrane on filter paper to airdry.
13. When using HRP color development substrate, wash each well twice with 200 µl TBS to
eliminate excess Tween 20. This wash step is not necessary when using AP systems or
NBT/BCIP color development. Add 100–200 µl of the color development solution to each
well. The reagent can be allowed to react while the solution slowly drains by gravity
filtration or the reaction time can be extended by closing the flow valve prior to adding the
substrate. In either application, when the color development is completed, the excess
substrate should be removed by vacuum and all the sample wells should be vacuum
washed with 200 µl of distilled water to stop the reaction. Following this wash step,
remove the membrane from the apparatus. Rinse the membrane in distilled water and
allow it to airdry on filter paper.
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