1. This is usually due to the filter plate being clogged.
If the filter plate is clogged, the clog may be dislodged under some circumstances by gently pushing the pointed end of a 15 mL plastic conical tube into the bottom of the plate under the clogged well. This procedure clears the small opening in the plastic casing. To prevent filter plate clogging, clarify samples by centrifugation at 14,000 x g for 30 seconds prior to analysis. Some samples may also require filtration prior to analysis. See Technote #49 for Sample Preparation Guidlines..

2. The lack of a tight seal between the plate and the manifold or a inadequate vacuum pressure could be the cause of the problem. Hold the plate firmly against the vacuum manifold to form a tight seal. If only a partial plate is being analyzed, cover the empty wells with a self adhesive plate seal. See Technote #50 for Millipore user guides