Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 9)

3.2 Helpful Hints
1. During the assay, do not leave the vacuum on. This may dehydrate the membrane and may
cause halos around the wells. Apply vacuum only until solutions are removed from the
sample wells, then adjust the flow valve so that the unit is exposed to air and disconnect the
vacuum source.
2. If some sample wells are not used in a particular assay, those wells must be closed off to
insure proper vacuum to the wells in use. There are three ways to close off unused wells.
One is to apply a 3% gelatin solution to those wells. Gelatin will clog the membrane and cut
off the vacuum flow to the clogged wells. The second method is to cover the unused portion
of the apparatus with tape to prevent air from moving through those wells. The third method
is to add buffer to the empty wells at each step instead of sample or wash solutions.
3. Any particulate in samples or solutions will block the membrane and restrict flow of solutions
through the membrane. For best results, filter or centrifuge samples to remove particulate matter.
4. Check the wells after sample has been applied to insure that there are no air bubbles in the
wells. Air bubbles will prevent the sample from binding to the membrane. Air bubbles may be
removed by pipetting the liquid in the well up and down.
5. Proper positioning of the flow valve relative to the level of the apparatus is important for
proper drainage. The speed of filtration is determined by the difference in hydrostatic
pressure between the fluid in the sample wells and the opening of the flow valve which is
exposed to air. If the opening of the flow valve is above the level of the sample wells very
little drainage will occur. When the flow valve is positioned where it is at a level below the
sample wells proper drainage will occur during filtration applications.
6. The recommended sample loading volume is at least 200 µl. If sample volumes of less than
200 µl are loaded, they must be carefully applied to the center of the well. Applying the
solution on one side of the well results in unequal distribution of sample. This results in
unevenly shaped bands, leading to distorted densitometer readings.
7. The Bio-Dot SF apparatus is designed for use with an 8-channel pipet allowing eight sample
or wash solutions to be quickly and easily applied to one row at a time.
8. The best method for removing the blotted 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.
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