I experienced, that when I used 3 or 5 kd cut-off filter tubes to exchange buffer for proteomics work, the flow through have some protein content. Theoretically it should not have happened. In contrast, I detected proteins in the flow through by bradford assay. I am afraid that I lost some proteins during buffer exchange before processing.
Do you use concentration devices (via centrifugation) or dialysis with tubings (some people call it membrane)? Depending on the protein size and its stability one can successfully use concentration devices (I believe that is what you call "cut-off filter tubes") to rapidly change buffer. The procedure, unfortunately, may backfire if the protein is not stable, conditions are not correctly chosen (salt concentration, salt type, pH, presence of divalent ions, presence of DTT or b-ME) or the sample is over-concentrated. All that may lead to protein loss. In your case, if you see the protein in the flow-through and it is not supposed to be there due to the size exclusion, check on the membrane. In my experience, upon long storage (especially at relatively high temperature or when exposed to direct light ) the membrane in low kDa devices may become detached and sometimes even perforated . That would explain why you see your protein in flow-through.
Good luck
I used membrane filterers by centrifuge. I also used reported replacing buffer from the literature with according to manufacturer protocol for labeling. I received new columns recently and stored at appropriate temp.
Usually manufacturers of filters with a cutoff o x kd, indicate that with a sample with that molecular mass, you will have a recovery of around 90%. Try to have a nice gap between your sample of interest and the filter cutoff. As previously said, you may also be seeing either smaller contaminants or be working at centrifugal forces higher than the recommended in each device.
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