Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 10)

Section 4
Protein Slot Blotting
4.1 General Recommendations
1. Solution Volume.
The liquid in the incubation vessel should be least 0.25 cm deep to ensure the membrane is
completely submerged during incubation. There should be at least 0.5 ml of reagent per cm
2
of membrane. Larger volumes may be used for convenience.
2. Handling the mMembrane.
Wear clean plastic gloves or use forceps to avoid fingerprints on the membrane.
3. Temperature.
All steps are performed at room temperature (22–25°C).
4. Incubation Vessels.
Incubation vessels may be made of plastic or glass. However, since avidin binds to
unsiliconized glass, plastic or siliconized glass vessels should be used whenever biotin-avidin
systems are employed for detection.
5. Membrane Incubation.
Agitation with a rotating shaker platform enhances incubation efficiency. If a shaker platform
is not available, hand mixing every few minutes and extended incubation periods will suffice.
6. Detection.
It is best to incubate only one membrane per vessel. Should it become necessary to use
more than one membrane per incubation vessel, calculate the solution volume based on the
membrane surface area, not the vessel size.
4.2 Immunoassay Procedure
Detailed instructions, including a comprehensive troubleshooting guide, for performing
immunoassays are given in the Immun-Blot ® instruction manuals.
1. Assemble the Bio-Dot SF apparatus as described in Section 3.1. Prewet the membrane prior
to placing it in the apparatus. Nitrocellulose membranes are prewetted in TBS; Zeta-Probe
membrane is prewetted in distilled water (see Section 10 for solution preparation). Make sure
that all the screws have been tightened under vacuum to ensure that there will not be any
cross-well contamination.
2. Rehydrate the membrane to ensure uniform binding of the antigen. Use 100 µl TBS per well
for nitrocellulose membranes. Use 100 µl distilled water per well for the Zeta-Probe
membrane.
3. Adjust the flow valve so that the vacuum chamber is open to air (flow valve setting 2, Figure
3). Fill the appropriate wells with antigen (protein) solution, applying 50–500 µl per well. The
recommended sample loading volume is at least 200 µl. If less than 200 µl is applied, the
sample 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.
Note: The solution applied should be free of insoluble particles to avoid clogging of wells.
4. Allow the entire sample to filter through the membrane by gentle vacuum. Make sure that the
flow valve is positioned at a level below the sample wells to ensure proper drainage during
filtration applications. Slow, gentle filtration is necessary for quantitative antigen binding.
Each well should be filled with the same volume of sample solution to ensure homogeneous
filtration of all sample wells.
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